Methods of treating progressive forms of multiple sclerosis

ABSTRACT

The present disclosure relates to methods of treating individuals suffering from progressive forms of multiple sclerosis.

1. CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of provisional application No. 61/521,343 and provisional application no. 61/521,338, both of which were filed Aug. 8, 2011, and are incorporated herein by reference in their entireties.

2. SEQUENCE LISTING

This application contains a Sequence Listing which has been submitted in ASCII format via EFS and is hereby incorporated by reference. The ASCII copy, created on Aug. 8, 2012, is named 240WO.TXT and is 34,203 bytes in size.

3. BACKGROUND

Daclizumab (DAC) is a humanized IgG₁ monoclonal antibody that binds to the alpha subunit (CD25 or Tac) of the human high-affinity interleukin-2 (IL-2) receptor, which is expressed on the surface of activated, but not resting, T- and B-lymphocytes. When bound to CD25 on the activated cells, DAC blocks the formation of the high affinity IL-2 receptor complex, thereby blocking IL-2-induced proliferation of the activated cells.

As measured in direct binding assays on PHA blasts, DAC binds to CD25 with an approximate binding affinity (K_(D)) of 0.3 nM, and inhibits the proliferation of PHA blasts in a dose-dependent manner (Hakimi et al., 1993, J. Immunol. 151(2):1075-85). At a suboptimal dose of IL-2 (2.5 ng/mL), 15 nM DAC inhibits proliferation of IL-2-dependent cell line Kit225/K6 by 50% (Pilson et al., 1997, J. Immunol. 159(3):1543-56). In an IL2-dependent antigen-induced T-cell proliferation assay, 50% inhibition of proliferation was observed with DAC in the range of 0.5-1 μg/mL (3-7 nM) (Junghans et al., 1990, Cancer Res. 50(5):1495-1502).

A version of DAC was previously marketed for the treatment of acute allograft rejection in renal transplant patients as an adjunct to an immunosuppressive regimen that includes cyclosporine and corticosteroids by Hoffman-La Roche, Inc. under the tradename ZENAPAX™. ZENAPAX was supplied as a concentrate for further dilution and intravenous administration. Each vial of concentrate contained 5 mL of a solution containing 5 mg/mL DAC, 3.6 mg/mL sodium phosphate monobasic monohydrate, 11 mg/mL sodium phosphate dibasic heptahydrate, 4.6 mg/mL sodium chloride, 0.2 mg/mL polysorbate 80 and HCl and/or NaOH sufficient to adjust the pH to pH 6.9. The recommended dose for both adult and pediatric patients was 1.0 mg/kg, prepared by diluting the calculated volume of 25 mg/5 mL ZENAPAX concentrate with 50 mL sterile 0.9% sodium chloride solution and administering intravenously via a peripheral or central vein over a 15-minute period.

DAC has also shown efficacy in the treatment of uveitis (Nussenblatt et al., 2004, FOCIS 2004 meeting; Jul. 18-23, Montreal, QC. Abstract 4688; Nussenblatt et al., 2003, J. Autoimmun. 21:283-93) and multiple sclerosis (see, e.g., Bielekova et al., 2004, Proc. Nat'l. Acad. Sci. USA 101(23):8705-8708; Rose et al., 2007, Neurology 69:785-789; U.S. Pat. No. 7,258,859), and is currently the subject of ongoing clinical trials for the treatment of relapsing forms of multiple sclerosis. Currently, there are few, if any, treatments available for progressive forms of multiple sclerosis. The availability of such treatments would be desirable.

4. SUMMARY

As mentioned in the Background Section, daclizumab is a humanized IgG₁ antibody that specifically binds the alpha subunit (also referred to as CD25 or Tac) of the human interleukin-2 receptor (IL-2R), which is an important mediator of lymphocyte activation. A version of daclizumab previously marketed by Hoffman-La Roche under the tradename ZENAPAX™ has demonstrated safety and efficacy in the treatment of renal allograft rejection when used as an adjunct to an immunosuppressive regimen including cyclosporine and corticosteroids (see, e.g., the European Medicines Agency (“EMEA”) market authorization for ZENAPAX), and has also demonstrated efficacy in the treatment of relapsing forms of multiple sclerosis (see, e.g., Bielekova et al., 2004, Proc. Nat'l. Acad. Sci. USA 101(23):8705-8708; Rose et al., 2007, Neurology 69:785-789; U.S. Pat. No. 7,258,859).

Clinical investigations studying the efficacy of a version of daclizumab called “DAC HYP” in relapsing and remitting multiple sclerosis (“RRMS”) are currently ongoing. One recently completed double-blinded placebo-controlled study, called the SELECT study, had two treatment arms: (i) a “low dose” arm in which patients received 150 mg DAC HYP subcutaneously once every four weeks; and (ii) a “high dose” arm in which patients received 300 mg DAC HYP subcutaneously once every four weeks. Fifty two week data from this SELECT study revealed surprising results—a statistically significant reduction in the risk of disability progression as measured by EDSS at one year. The reduction in EDSS was observed in both treatment arms, and the magnitude of the decrease observed after one year of treatment is unprecedented. Moreover, the effect of DAC HYP on disability progression was mediated by a reduction in the proportion of patients with disabling relapses in the placebo-treated groups versus DAC HYP-treated groups and by a reduction in disability progression that was independent of relapse. These results indicate that daclizumab can be used to treat relapsing forms of MS, forms or stages of MS in which patients are not experiencing relapses or exacerbations, and also as progressive forms of MS, including both primary progressive and secondary progressive forms of MS.

Accordingly, in one aspect, the present disclosure provides methods of treating human subjects suffering from progressive forms of multiple sclerosis. The method generally involves administering to a human subject suffering from a progressive form of MS an amount of a daclizumab effective to provide therapeutic benefit, for example, in some specific embodiments an amount effective to reduce the risk of disability progression as assessed by EDSS by at least about 50% approximately one year after initiation of therapy. The progressive form of MS can be secondary progressive, primary progressive or other progressive form of MS.

In another aspect, the present disclosure provides methods of treating patients who suffer from a form or stage of MS in which the patients are not experiencing relapses or exacerbations. The patient may be diagnosed with relapsing-remitting MS that may be in a stage of disease in which s/he has not suffered a relapse or exacerbation for an extended period of time, for example a period of 6 months, 9 months, 12 months, 18 months, 24 months or even longer, or may be diagnosed with secondary progressive MS that has evolved into the non-relapsing progressive stage, or may be diagnosed with progressive-relapsing MS. The method generally involves administering to a human subject suffering from a form or stage of MS in which the subject has not experienced a relapse or exacerbation for a period of at least about 6, 9, 12, 18 or 24 months, and in some specific embodiments a period of at least about 12 months, an amount of a daclizumab effect to provide therapeutic benefit, for example, is some specific embodiments an amount effect to reduce the risk of disability progression as assessed by EDSS by at least 50% approximately one year after initiation of therapy.

Effective amounts typically range from about 150 mg to about 300 mg, inclusive, administered subcutaneously once every four weeks for a period of time sufficient to confer therapeutic benefit, for example a period of at least one year.

The 52-week SELECT results also revealed certain liver function abnormalities, manifested by levels of aspartate aminotransferase (AST) and/or alanine aminotransferace (ALT) that are elevated above normal levels. Accordingly, in another aspect, the present disclosure provides methods of monitoring patients receiving daclizumab therapy, for example, DAC HYP therapy, for liver enzyme abnormalities. The method generally comprises determining the level of AST and/or ALT in a patient receiving daclizumab therapy to assess whether the treatment should be discontinued or modified. Guidance for making specific treatment decisions based upon the levels measured is provided in the Detailed Description section.

5. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides DAC-HYP light chain cDNA (SEQ ID NO: 1) and translated amino acid (SEQ ID NO:2) sequences. The bold, underlined aspartate (D) residue is the first amino acid in the properly processed mature protein; the amino acid sequence upstream of this residue corresponds to the signal sequence.

FIG. 2 provides DAC-HYP heavy chain cDNA (SEQ ID NO:3) and translated amino acid sequences (SEQ ID NO:4). The bold, underlined glutamaine (Q) residue is the first amino acid in the properly processed mature protein; the amino acid sequence upstream of this residue corresponds to the signal sequence.

FIG. 3A-FIG. 3D together provide the full nucleotide sequence for vector pHAT.IgG1.rg.dE (SEQ ID NO:5).

FIG. 3E provides a specific embodiment of a dESV40 promoter (SEQ. ID NO: 12) that can be used to select high yielding producer strains.

FIG. 4A-4B provide a schematic diagram of vector pHAT.IgG1.rg.dE (FIG. 4A), which is derived from pABX.gpt, a vector that can be adapted to express any heavy and light chain genes or even a non-antibody polypeptide (FIG. 4B).

FIG. 5 provides an exemplary production process for DAC HYP.

FIG. 6 demonstrates UV (280 nm), pH and conductivity monitoring of product fractions during protein A affinity chromatography.

FIG. 7 demonstrates UV (280 nm), pH and conductivity monitoring of product fractions during Q-sepharose chromatography.

FIG. 8 demonstrates UV (280 nm), pH and conductivity monitoring of product fractions CM cation exchange chromatography.

FIG. 9 provides a schematic illustration of the DAC HYP ultrafiltration system.

FIG. 10 provides a 0-60 minute DAC HYP peptide map chromatogram. The reference profile is a 100 mg/ml DAC HYP preparation and Batch 1 and Batch 2 correspond to 150 mg/ml DAC HYP preparations.

FIG. 11 provides a 55-115 minute DAC HYP peptide map chromatogram. The reference profile is a 100 mg/ml DAC HYP preparation and Batch 1 and Batch 2 correspond to 150 mg/ml DAC HYP preparations.

FIG. 12 provides a 110-170 minute DAC HYP peptide map chromatogram. The reference profile is a 100 mg/ml DAC HYP preparation and Batch 1 and Batch 2 correspond to 150 mg/ml DAC HYP preparations.

FIG. 13 provides overlaid circular dichroism spectra of DAC HYP 150 mg/ml lots Batch 1 and Batch 2. The reference is a 100 mg/ml preparation of DAC HYP.

FIG. 14A-FIG. 14B provide overlaid zero-order ultraviolet spectra and overlaid second derivative ultraviolet spectra, respectively. The reference profile is a 100 mg/ml DAC HYP preparation and Batch 1 and Batch 2 correspond to 150 mg/ml DAC HYP preparations. All three spectra are present in each of FIG. 4A and FIG. 4B, but appear as a single spectrum when overlaid on one another.

FIG. 15A-FIG. 15B provide full scale and expanded scale size exclusion chromatograms, respectively. The reference profile is a 100 mg/ml DAC HYP preparation and Batch 1 and Batch 2 correspond to 150 mg/ml DAC HYP preparations.

FIG. 16 is a plot of DAC HYP aggregation as a function of time.

FIG. 17 shows reduced and non-reduced SDS-PAGE (left and right panels, respectively). The reference profile is a 100 mg/ml DAC HYP preparation and Batch 1 and Batch 2 correspond to 150 mg/ml DAC HYP preparations.

FIG. 18 shows cation exchange chromatograms of DAC HYP. The reference profile is a 100 mg/ml DAC HYP preparation and Batch 1 and Batch 2 correspond to 150 mg/ml DAC HYP preparations. The peak labels correspond to the different N- and C-terminal isoforms.

FIG. 19 shows HPLC chromatograms of N-linked oligosaccharides enzymatically cleaved from DAC HYP. The reference profile is a 100 mg/ml DAC HYP preparation and Batch 1 and Batch 2 correspond to 150 mg/ml DAC HYP preparations.

FIG. 20 shows ADCC response curves of DAC HYP. The reference profile is a 100 mg/ml DAC HYP preparation and Batch 1 and Batch 2 correspond to 150 mg/ml DAC HYP preparations.

FIG. 21 shows HPLC chromatograms for N-linked oligosaccharides released from DAC HYP (lower panel) and ZENAPAX DAC (upper panel) illustrating their different glycosylation profiles.

FIG. 22A-FIG. 22B provide a comparison between the ADCC activity of two DAC HYP preparations (referred to as DAC HYP Batch 3 and DAC HYP Batch 4), DAC Penzuburg, and ZENAPAX DAC using the variable effector-to-target cell ratio ADCC assay format (FIG. 22A) and the variable antibody concentration ADCC assay format (FIG. 22B).

FIG. 23 provides a comparison of the charge isoforms of DAC HYP, DAC Penzberg and ZENAPAX DAC.

6. DETAILED DESCRIPTION

The present disclosure provides methods of treating patients suffering from progressive forms of multiple sclerosis as well as patients suffering from forms or stages of MS in which the patient has not experienced a relapse or exacerbation for a significant period of time, for example a period of at least 6 months. Multiple sclerosis (“MS”) is generally understood to be an autoimmune disease clinically described as a central nervous system white matter disorder disseminated in time and space. It can be diagnosed by brain and spinal chord magnetic resonance imaging (“MRI”), analysis of somatosensory evoked potentials, and analysis of cerebrospinal fluid to detect increased amounts of immunoglobulin or oligoclonal bands. MRI is a particularly sensitive tool. MRI abnormalities indicating the presence or progression of MS include hyperintense white matter signals on T2-weighted and fluid attenuated inversion recovery images, gadolinium enhancement of active lesions, hypointensive “black holes” (representing gliosis and axonal pathology) and brain atrophy on T1-weighted studies.

MS presents in several forms, generally characterized by two main forms: relapsing forms and progressive forms. In relapsing forms, new symptoms tend to occur in diserete attacks. In progressive forms, new symptoms accumulate progressively over time (see, e.g., Lublin & Reingold, 1996 Neurology 46(4):907-911)). The four currently recognized forms of MS and their main characteristics are:

-   -   Relapsing-remitting MS: characterized by unpredictable acute         attacks, called “exacerbations,” with worsening of symptoms         followed by full, partial, or no recovery of some function.         These attacks appear to evolve over several days to weeks.         Recovery from an attack takes weeks sometimes months. The         disease does not worsen in the periods between the attacks. This         pattern usually occurs early in the course of MS in most people.     -   Primary-progressive MS: characterized by a gradual but steady         progression of disability, without any obvious relapses and         remissions. This form of disease occurs in just 15% of all         people with MS, but it is the most common type of MS in people         who develop the disease after the age of 40.     -   Secondary-progressive MS: initially begins with a         relapsing-remitting course, but later evolves into progressive         disease. The progressive part of the disease may begin shortly         after the onset of MS, or it may occur years or decades later.     -   Progressive-relapsing MS: This is the least common form of the         disease and is characterized by a steady progression in         disability with acute attacks that may or may not be followed by         some recovery. People with progressive relapsing MS initially         appear to have primary progressive MS.

The present disclosure concerns the treatment of progressive forms of MS, including both primary progressive and secondary progressive, as well as forms or stages of MS that are characterized by a lack of relapses or exacerbations. Diagnostic criteria for assessing whether an individual is suffering from primary progressive MS or secondary progressive MS are well-known. For example, for primary progressive MS (“PPMS”), three levels of diagnostic certainty have been defined—definite, probable and possible-based on clinical findings, abnormal cerebrospinal fluid, abnormalities on MRI of the brain and special cord and evoked potentials. According to one set of criteria, in definite PPMS, evidence of intrathecal synthesis of immunoglobulin G together with one of the following three MRI criteria is indicitative of PPMS: (1) nine brain lesions, (2) two spinal cord lesions, (3) four to eight brain lesions and one spinal cord lesion (see, e.g., Thompson et al., 2000, Ann. Neurol. 47(6):831-835). These and other well-established diagnostic criteria can be used to diagnose an individual as having PPMS. Ultimate diagnosis will depend upon the judgment of the neurologist.

Diagnosis of individuals as having secondary progressive MS (“SPMS”) is largely based upon the judgment of the neurologist. Essentially, when an individual with RRMS becomes aware of a definite deterioration in their level of function that continues for at least six months to one year which is not related to a relapse, then it is likely that the individual now has SPMS. This is often common in individuals who already manifest some problems or disability as a result of previous MS relapses that have not recovered completely.

Guidance for diagnosing patients can also be found in McDonald et al., 2001, Ann. Neurol. 50(1):121-127.

Data presented herein demonstrate that treatment of individuals having MS with a daclizumab reduces disability progression in a manner that is independent of relapse. These data indicate that individuals suffering from relapsing-remitting forms of MS will receive significant benefit from continuing daclizumab therapy even during periods of significant remission from relapses or exacerbations, for example periods of 6 months, 12 months, 18 months or even longer.

The methods generally involve administering to the subject an amount of daclizumab effective to provide therapeutic benefit. Therapeutic benefit may be assessed in numerous ways, as is known in the art. In specific embodiments, therapeutic benefit is achieved when there is a measurable decrease in the rate of the patients' disability progression. In specific embodiments, patients are administered an amount of a daclizumab effective to reduce the average increase in the subject's disability score by at least about 50% over a period of one year, as measured by the EDSS score. For example, the DAC compositions described herein can be administered subcutaneously to a patient from weekly to monthly (e.g., weekly, every two weeks, twice a month, every four weeks or monthly) in doses ranging from 75 mg to 300 mg (e.g., 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg or 300 mg) or ranging from 1 mg/kg to 4 mg/kg. The compositions can be provided in prefilled syringes convenient for subcutaneous use, preferably at nominal daclizumab concentrations of 100 mg/mL+10-15% or 150 mg/mL+10-15%. The concentrated DAC compositions can also be diluted for intravenous administration. It has been found from the SELECT study that administering from about 150 mg up to about 300 mg daclizumab subcutaneously once every four weeks for a period of one year, provides therapeutic benefit.

While the SELECT study utilized a specific version of daclizumab called DAC HYP, the methods described herein are not limited to a specific version or formulation of daclizumab. It is expected that any daclizumab or daclizumab analog will be effective.

Daclizumab (DAC) as used herein refers to a humanized IgG₁ monoclonal antibody having the CDRs of the variable light (V_(L)) chain sequence illustrated in FIG. 1 (positions 21-233 of SEQ ID NO:2) and CDRs of the variable heavy (V_(H)) chain sequence illustrated in FIG. 2 (positions 20 to 465 of SEQ ID NO:4). The CDR sequences of DAC are as follows:

V_(L)CDR#1: (SEQ ID NO: 6) S A S S S I S Y M H V_(L)CDR#2: (SEQ ID NO: 7) T T S N L A S V_(L)CDR#3: (SEQ ID NO: 8) H Q R S T Y P L T V_(H)CDR#1: (SEQ ID NO: 9) S Y R M H V_(H)CDR#2: (SEQ ID NO: 10) Y I N P S T G Y T E Y N Q K F K D V_(H)CDR#3: (SEQ ID NO: 11) G G G V F D Y

In a specific embodiment, the daclizumab may be that described in U.S. Pat. No. 5,530,101 (see, e.g., SEQ ID NO:5 setting forth the variable heavy chain sequence and SEQ ID NO:7 setting forth the variable light chain sequence). A version of daclizumab was previously marketed by Roche under the trade name ZENAPAX. Another version of a daclizumab was produced by Roche in Penzberg, Germany. These versions of daclizumab, both of which can be used in the methods described herein, are referred to as DAC ZENAPAX and DAC Penzberg, respectively. In a specific embodiment, the daclizumab may be a DAC HYP, which is described in more detail below and in the Exemplary Embodiments, as are various tools, constructs and methods for making a DAC HYP.

Generally, DAC HYP compositions are characterized by one or more of the following features:

(1) a characteristic pE/Q and/or Q/VHS N-terminal isoform;

(2) a homogeneous N-linked oligosaccharide profile characterized by two main peaks and a minor peak;

(3) reduced ADCC toxicity as compared to ZENAPAX DAC and/or DAC Penzberg; and

(4) a low level of aggregate forms (<3%) when formulated a nominal concentrations as high as 150 mg/ml (±10-15%).

DAC compositions having one or more of these characteristics and/or properties (discussed further below) are referred to herein as “DAC HYP” compositions. For purposes of exemplifying the various aspects and features of inventions described herein, a specific DAC HYP having all four of the above properties is described in the Exemplary Embodiments, as are specific compositions and methods for its production and purification. However, it is to be understood that a DAC HYP composition need not have all of the above four characteristics. In specific embodiments, a DAC HYP has at least two of characteristics (1) through (4) above (e.g., at least a combination of (1) and (2); (1) and (3); (1) and (4); (2) and (3); (2) and (4); or (3) and (4)) or at least three of characteristics (1) through (4) above (e.g., at least a combination of (1), (2) and (3); (1), (2) and (4); (1), (3), and (4); and (2), (3), (4)). Such DAC HYP compositions can also have <3% aggregates, <2% aggregates and even lower levels, e.g., <1% aggregates, when formulated at concentrations of 100 mg/ml (+10-15%) or even 150 mg/ml (±10-15%).

Isoforms of daclizumab are possible due to heterogeneity at the heavy chain N- and C-termini. The amino acid sequence of the mature V_(H) chain of daclizumab begins at position 20 of the amino acid sequence shown in FIG. 2 (SEQ ID NO:4). The N-terminal glutamine (Q) of the mature V_(H) chain (in bold, underlined text in FIG. 2) can cyclize, forming pyroglutamate (pE). In some instances, the signal peptide sequence may truncate, leaving a valine-histidine-serine (VHS) sequence attached to the N-terminal glutamine residue of the mature V_(H) chain. Because each daclizumab molecule contains two V_(H) chains, the various N-terminal isoforms of daclizumab can include forms containing: (1) two glutamine residues (Q/Q); (2) one glutamine residue and one VHS sequence (Q/VHS or VHS/Q); (3) two VHS sequences (VHS/VHS); (4) one glutamine residue and one pyroglutamate residue (Q/pE or pE/Q); (5) one pyroglutamate residue and one VHS sequence (pE/VHS or VHS/pE); and (6) two pyroglutamate residues (pE/pE). Different C-terminal isoforms also are possible, which contain either 0, 1 or 2 C-terminal lysine (K) residues (0K, 1K or 2K), resulting in a complex isoform profile.

While the N-terminal glutamines of the V_(H) chains of ZENAPAX DAC are completely cyclized to pyroglutamate, complete cyclization is not achieved for DAC HYP.

As a consequence, the cation exchange chromatogram of DAC HYP is characterized by a pE/Q isoform peak and a Q/VHS isoform peak. While not intending to be bound by any theory, it is believed that these unique pE/Q and Q/VHS isoforms may be influenced by the leader sequence used to express DAC HYP. In certain embodiments, DAC HYP is characterized by having a pE/Q isoform that ranges from 3%-17%, from 3%-15%, from 5%-15%, more preferably from 5%-12% or 7%-12% of the N-terminal isoforms, and/or in which the Q/VHS isoform ranges from 1%-15%, more preferably 3%-12% of the N-terminal isoforms, as determined by cation exchange chromatography.

In some specific embodiments, a DAC HYP composition is characterized by a cation exchange chromatography profile that is substantially similar to FIG. 18 or the DAC HYP profile of FIG. 23.

Daclizumab has N-linked oligosaccharides attached to heavy chain residue Asn 296. When these N-linked oligosaccharides are released using amidase PNGaseF and analyzed via HPLC, DAC HYP exhibits a glycosylation profile different from ZENAPAX DAC, despite the fact that both are recombinantly produced in NS0 cell lines. Indeed, the glycosylation profile of DAC HYP is unusually homogeneous. Referring to the upper panel of FIG. 21, the glycosylation profile of ZENAPAX DAC is characterized by peaks representing oligosaccharides G0-GlcNAc, G0, G1, Man5, G2, Man6, Man7 and sialylated oligosaccharide. The lower panel of FIG. 21 shows that the glycosylation profile of DAC HYP is characterized by two main peaks corresponding to G0-GlcNAc glycoforms and G0 glycoforms and a minor peak corresponding to a G1 glycoform. The G0-GlcNAc glycoforms can range from about 5% to about 20% of the AUC, more typically about 7.2% to 14.6% of the AUC. The G0 glycoforms can range from 70% to 99.2% of the AUC, more typically from 80.9% to 99.2% of the AUC. The G1 glycoform can range from 1% to 9% of the AUC, more typically from 1.4% to 3.8% of the AUC. Sialylated oligosaccharides are 1.0% of the total AUC or less.

Immunogenicity and high levels of effector function can be problematic for chronically administered drugs. In addition, rapid clearance rates can reduce drug availability. As is well-known by skilled artisans, differences in glycosylation patterns of therapeutic antibodies can give rise to differences in immunogenicity. Antibodies having highly homogeneous glycosylation patterns like DAC HYP may provide beneficial immunogenicity profiles, ADCC levels, and clearance rates. In addition, biologics having more homogeneous glycosylation patterns reduce batch to batch variation and can improve consistency and stability.

Accordingly, in some embodiments, a DAC HYP composition is characterized by a homogeneous N-linked glycosylation profile. In one embodiment, a DAC HYP composition is characterized by an N-linked glycosylation profile that includes approximately 5-20% of the total AUC of G0-GlcNAc glycoforms, and in some embodiments approximately 5%-18% or approximately 7-15% (e.g., 7.2%-14.6% or 6.9%-14.7%) of the total AUC of G0-GlcNAC glycoforms (and in some specific embodiments 7.3% of the total AUC of G0-GlcNAc glycoforms), and approximately 70%-99.2% of the total AUC of G0 glycoforms, and in some embodiments approximately 75%-90%, approximately 75-92%, or approximately 81-88% of the total AUC of G0 glycoforms (and in some specific embodiments 86% of the total AUC of G0 glycoforms), as measured by HPLC. Optionally, the G1 peak is less than about 10% of the total AUC, less than about 5%, less than about 4% or less than about 3% of the total AUC and, in certain embodiments, ranges from about 1% to about 4% (e.g., 1.4% to 3.8%) or about 1% to about 3%. The Man5 glycoforms are preferably about 3% of the total AUC or less. In other embodiments, a DAC HYP composition is characterized by an HPLC N-linked glycoform profile substantially similar to a profile illustrated in FIG. 19.

In certain embodiments, a DAC HYP composition is characterized by the sum total of two or more glycoform peaks. For example, a DAC HYP composition may be characterized by (a) two main peaks corresponding to G0-GlcNAc glycoforms and G0 glycoforms which together range from about 75% to about 100%, from about 80% to about 100%, or about 85% to about 100% of the total AUC and/or (b) peaks corresponding to Man5, Man6, and Man7 glycoforms which together are about 6% of the total AUC or less and/or (c) peaks corresponding to Man6 and Man7 glycoforms which together are about 2% of the total AUC or less. In such embodiments, the percentage of G0-GlcNAc G0, G1, and/or Man5 can be present in the amounts described in the preceding paragraph.

The binding and inhibitory properties of DAC HYP, as well as the functional potency of DAC HYP as evaluated in an assay that measures the inhibition of IL-2-induced proliferation of T-cells, are similar to those of ZENAPAX DAC. However, DAC HYP exhibits significantly less ADCC cytotoxicity than ZENAPAX DAC, which is likely due, at least in part, to differences in their non-fucosylated mannose glycosylation levels (see FIG. 21). As shown in FIG. 22A and FIG. 22B, DAC HYP exhibits at least 25% less ADCC cytotoxicity than ZENAPAX DAC as measured in a cellular assay. As will be recognized by skilled artisans, the reduced ADCC cytotoxicity of DAC HYP may be beneficial for indications involving chronic administration where cell death is not desirable, for example, for the treatment of multiple sclerosis or uveitis. In these contexts, DAC HYP therapy may be safer than therapy with ZENAPAX™.

Accordingly, in some specific embodiments, DAC HYP compositions are characterized by exhibiting ADCC cytotoxicity of less than about 30%, 25%, 20%, 15%, 10%, 5%, or even lower, at a concentration of 1 μg/mL as measured in an in vitro assay using an effector to target cell ratio of 25:1, 40:1, 50:1 or 60:1, for example when using Kit225/K6 as a target cell and/or when using PBMC effector cells from 3 or more, 6 or more, 10 or more, or 50 or more healthy donors. In specific embodiments, DAC HYP compositions are characterized by exhibiting ADCC cytotoxicity ranging from 5-30%, from 10-30%, from 15-30%, from 15-30%, from 5-25%, from 10-25%, from 20-30%, from 15-25%, from 15-35%, or from 20-35% at a concentration of 1 μg/mL as measured in an in vitro assay using an effector to target cell ratio of 25:1, 40:1, 50:1 or 60:1, for example when using Kit225/K6 as a target cell and/or when using PBMC effector cells from 3 or more, 6 or more, 10 or more, or 50 or more healthy donors.

Daclizumab compositions characterized by one or more of the properties discussed above (DAC HYP compositions) can be conveniently obtained via recombinant expression in mammalian cells. While not intending to be bound by any particular theory of operation, it is believed that one or more of the characteristics and/or properties discussed above may be due, at least in part, to the use of a high productivity recombinant expression system. This can be achieved by any method, such as by gene amplification using the DHFR, or using a selectable marker gene under the control a weak promoter, preferably in combination with a strong promoter driving the expression of the protein of interest (preferably a secreted protein). Without being bound by theory, it is believed that selection of markers under the control of a weak promoter facilitates the identification of stable transfectants in which the expression vector integrates into a chromosomal region that is transcriptionally active, yielding high expression levels of the protein of interest. In one embodiment, the weak promoter driving the expression of a selectable marker is an SV40 promoter (Reddy et al., 1978, Science 200:494-502) in which the activity of one or both enhancer regions has been reduced or eliminated, such as by partial or complete deletion (designated “dESV40”), optionally in combination with a strong promoter, such as the CMV IE promoter (Boshart et al., 1985, Cell 41(2):521-30), driving expression of the protein of interest.

A specific dESV40 promoter sequence that can be used to produce stable expression cell lines is at positions 6536-6735 of vector pHAT.IgG1.rg.dE (SEQ ID NO:5), illustrated in FIG. 3A-FIG. 3D, and in FIG. 3E (SEQ ID NO: 12).

Generally, vectors useful for expressing a daclizumab such as DAC HYP will include one or more of the features exemplified by pHAT.IgG1.rg.dE (described in Section 7.1 below), such as a promoter. The two chains of daclizumab can be placed under separate transcriptional control but are preferably on the same vector, and their coding regions can be cDNA or genomic DNA containing introns and exons. As an alternative to separate transcriptional control, the two chains can be expressed as a single transcript or a single open reading frame, with their coding regions separated by an internal ribosome entry site or a self-cleaving intein sequence, in which case the heavy and light chain coding sequences are under the control of a single promoter. An exemplary promoter is the CMV IE promoter and enhancer (at positions 0001-0623 and 3982-4604 of pHAT.IgG1.rg.dE (SEQ ID NO:5)). Additional features include transcriptional initiation sites (if absent from the promoter chosen), transcription termination sites, and origins of replication. Examples of such features are illustrated in Table 1, which outlines the components of pHAT.IgG1.rg.dE.

A specific embodiment useful for expressing both heavy and light chains of a daclizumab such as DAC HYP from a single exogenous nucleic acid in NS0 cells utilizes a selection marker operable in mammalian cells, such as neomycin phosphotransferase (neo^(r)), hygromycin B phosphotransferase (hyg^(r)), hygromycin B phosphotransferase (Hph), puromycin-N-acetyltransferase (puro^(r)), blasticidin S deaminase (bsr^(r)), xanthine/guanine phosphoribosyl transferase (gpt), glutamine synthetase (GS) or Herpes simplex virus thymidine kinase (HSV-tk). In a specific embodiment, the selectable marker is an E. coli guanine phosphoribosyl transferase selectable marker under the control of an enhancer-less SV40 promoter, the encoding sequence of which can be found at positions 6935-7793 of pHAT.IgG1.rg.dE (SEQ ID NO:5) shown in FIG. 3A-FIG. 3D.

Host cells useful for producing a daclizumab recombinantly include mammalian cells, for example, Chinese Hamster Ovary (CHO) cells, NS0 murine myeloma cells, Sp2/0 cells, PER.C6 cells, Vero cells, BHK cells, HT1080 cells, COS7 cells, W138 cells, CV-1/EBNA cells, L cells, 3T3 cells, HEPG2 cells, MDCK cells and 293 cells. Once transfected, the vector may integrate into the genome to yield a stable production cell line. Skilled artisans will appreciate that it is undesirable to include animal products in compositions designated for administration to humans. Accordingly, host cells that do not require serum or other animal products for growth (such as, e.g., cholesterol) are preferred. Host cells that require such animal products can be adapted to utilize serum-free and other animal product-free medium. A method for adapting murine myeloma NS0 cells to grow in serum- and cholesterol-free medium is described in Hartman et al., 2007, Biotech. & Bioeng. 96(2):294-306 and Burky et al., 2007, Biotech. & Bioeng. 96(2):281-293. A specific strain of NS0 cells adapted to grow in serum-free and cholesterol-free medium that has been stably transfected with a vector as described above that can be used to produce daclizumab, and in particular a DAC HYP (clone 7A11-5H7-14-43, also referred to as Daclizumab dWCB IP072911) has been deposited with the American Type Culture Collection (ATCC), under accession no. ______.

The basal and feed media used to culture cells for recombinant protein production, as well as other variables such as the feeding schedule, growth rate, temperature, and oxygen levels, can affect the yield and quality of the expressed protein. Methods of optimizing these conditions are within the purview of a skilled artisan; exemplary conditions are set forth in the Exemplary Embodiments of the disclosure. In specific embodiments, cells are adapted to grow in media free of cholesterol-, serum-, and other animal-sourced components; in such instances the basal and feed media preferably include defined chemicals that substitute for such components. It has been discovered that media containing high levels of glucose, e.g., 10-35 g/L glucose, advantageously increase the cell culture productivity. In a specific embodiment, the basal medium has about 10-20 g/L, more preferably about 15 g/L, glucose and/or the feed medium has 22-35 g/L, more preferably around 28 g/L, glucose. The feed medium can be added to the cells according to an escalating feed schedule, as is known in the art, over a period of 8-15 days, 9-13 days, or, most preferably, 10-13 days.

For a DAC HYP expressed in NS0 producer strain 7A11-5H7-14-43, the components of the growth and feed media, and other variables affecting expression and production have been optimized. These media and culturing parameters and methods are described in more detail in Section 7.3.

Purifying daclizumab from a cell culture utilizing a combination of certain chromatography steps yields purified daclizumab and DAC HYP drug substance compositions and liquid daclizumab and DAC HYP drug formulations that are shelf stable in liquid form at high concentrations, typically at nominal daclizumab or DAC HYP concentrations of at least about 100 mg/mL+10-15% and in some embodiments 150 mg/mL±10-15% (as measured by UV spectroscopy or refractive index).

The stable, high concentration daclizumab drug formulations are generally prepared by exchanging a concentrated daclizumab formulation with exchange buffer having an osmolality in the range of about 267-327 mOsm/kg (e.g., 270-310 mOsm/kg) and a pH in the range of about pH 5.8-6.2 at 25° C. (e.g., 5.9-6.1 at 25° C.) to yield an intermediate formulation, and then diluting the intermediate formulation with polysorbate dilution buffer to yield a stable, high concentration liquid formulation comprising of about 100 mg/mL+10% daclizumab (e.g., DAC HYP), and in some embodiments at least about 150 mg/mL daclizumab (e g., DAC HYP), as measured by UV spectroscopy or refractive index. The dilution buffer is the same as the exchange buffer, but includes about 0-10% (w/v) polysorbate 80, and is used in an amount such that the final, stable, high concentration daclizumab formulation has a calculated polysorbate 80 concentration (nominal concentration) in a range of 0.02-0.04%, in some embodiments about 0.03% (w/v). A variety of different buffering agents and excipients can be included in the exchange and dilution buffers to achieve an osmolality and pH within the defined ranges. A specific, non-limiting example of an exchange buffer suitable for formulating stable, high concentration liquid daclizumab and DAC HYP drug formulations contains about 40 mM succinate and about 100 mM NaCl and has a pH of about 6.0 at 25° C. A specific, non-limiting example of a dilution buffer suitable for use with this exchange buffer contains about 40 mM succinate, about 100 mM NaC1 and about 1% (w/v) polysorbate 80 and has a pH of about 6.0 at 25° C. The pH of the final formulation can be adjusted with acid or base to yield an actual pH of about 6.0 at 25° C.

The stable, high concentration liquid daclizumab formulations are characterized by a low level of aggregation, typically containing at least 95% monomer and less than 3% aggregates, sometimes less than 1.5% aggregates, and more usually greater than 99% monomer and less than 0.8% aggregates, as measured by size exclusion chromatography. Other purity characteristics of the high concentration liquid daclizumab drug formulations are described in more detail in Section 7.6.

The high concentration daclizumab drug formulations are also characterized by a long shelf life, being stable against greater than 5% degradation and formation of greater than 3% aggregates (as measured by SDS-PAGE and size exclusion chromatography, respectively) for periods of up to 54 months or longer, for example, for at least 5 years, when stored at 2-8° C., for periods of up to 9 months when stored under accelerated conditions (23-27° C./60±5% relative humidity) and for periods of up to 3 months when stored under stressed conditions (38-42° C./75±5% relative humidity).

High concentration DAC HYP formulations are characterized by a low level of aggregates. It has been discovered that concentrating daclizumab via ultrafiltration induces aggregates to form, which can result in a high concentration daclizumab drug formulation containing unacceptable (e.g., >3%) levels of aggregates. Accordingly, it is preferable to utilize a “polishing” step prior to concentrating the daclizumab drug substance to remove aggregates. The level of acceptable aggregates prior to concentration will depend upon the concentration of the daclizumab drug substance to be concentrated, the desired concentration in the final daclizumab drug formulation, and the acceptable level of aggregates in the final daclizumab drug formulation. For example, if a 150 mg/mL daclizumab formulation containing less than 3% aggregates is desired, and the daclizumab drug substance must be concentrated 10- to 30-fold (e.g., 20-fold) to achieve this finished daclizumab formulation, the daclizumab composition to be concentrated should contain <0.3% aggregates, preferably <0.2% aggregates, and preferably even lower levels, e.g., about 0.1% aggregates.

A variety of known techniques can be used to obtain a starting daclizumab drug substance composition containing acceptable levels of aggregates for concentration into concentrated daclizumab intermediate and final drug formulations as described herein, including, for example, strong cation exchange chromatography and hydrophobic interaction chromatography. It has been discovered that weak cation exchange chromatography reduces levels of aggregates of daclizumab compositions containing in the range of 4-12 mg/mL daclizumab and up to 2.5% aggregates to extremely low levels, typically to about 0.1% aggregates. The use of weak cationic exchange to remove aggregates is more environmentally friendly than hydrophobic interaction chromatography, which utilizes nitrogen containing solutions (such as ammonium sulfate solutions).

Certain methods of polishing daclizumab compositions to remove aggregates yield resulting polished compositions that generally contain about 4 to 15 mg/mL daclizumab, where 0.3% or less (e.g., 0.2% or less or 0.1% or less) is in aggregate form, as measured by size exclusion chromatography. The method generally involves passing a daclizumab composition containing about 4-10 mg/mL, typically about 8-9 mg/mL, and preferably about 8.5 mg/mL daclizumab and >0.5% aggregates over a weak cation exchange resin in a suitable buffer to adsorb to the daclizumab, and eluting the adsorbed daclizumab with an elution buffer. Useful weak cation exchange resins include, but are not limited to, CM-650M (Tosoh Biosciences), CM-Sepharose, CM-HyperD. The components of the equilibration, washing and elution buffers will depend upon the weak cation exchange resin used, and will be apparent to those of skill in the art. For CM-650M resin (Tosoh Biosciences, part Number 101392), an equilibration and wash buffer containing about 20 mM sodium citrate, pH 4.5 and an elution buffer containing 20 mM sodium citrate and 75 mM sodium sulfate, pH 4.5 works well. The flow rate used will depend upon the choice of resin and size of the column. For a cylindrical column of CM-650M resin having a bed height in the range of about 10-30 cm (e.g., 17-19 cm) and a flow rate in the range of about 50-200 cm/hr (e.g., 90-110 cm/hr, preferably about 100 cm/hr), works well with the chromatography can be carried out at room temperature, or at lower temperature, for examples temperatures ranging from 4°, 10°, 15°, 20° or 25° C. A typical useful temperature range is 18-25° C. (e.g., 18-22° C.).

According to the ZENAPAX EMEA, the purification process for ZENAPAX DAC involves the following twelve steps:

(i) culture broth concentration;

(ii) Q-Sepharose chromatography;

(iii) S-Sepharose chromatography;

(iv) low pH treatment for viral inactivation;

(v) concentration/diafiltration;

(vi) DV50 filtration for virus removal;

(vii) Q-Sepharose II chromatography;

(viii) viresolve chromatography for virus removal;

(ix) concentration by ultrafiltration;

(x) S-300 gel filtration chromatography;

(xi) concentration by ultrafiltration;

(xii) aseptic filling of vials.

This process is inefficient, and provides a low purification yield. It has been discovered that higher yields can be achieved with a process having fewer steps, while at the same time yielding a higher degree of purity, which permits the resultant daclizumab drug substance to be formulated into high concentration drug formulations as described above. The process utilizes Protein A affinity chromatography in conjunction with strong anion exchange (Q-Sepharose) chromatography and weak cation exchange (CM-650M) chromatography, permitting continuous flow processing without dilution of process intermediate. The improved method for obtaining purified daclizumab drug substance involves the following steps:

(i) protein A affinity chromatography to isolate daclizumab from other cell culture components;

(ii) low pH viral inactivation;

(iii) strong anion exchange (Q-Sepharose) chromatography to remove DNA;

(iv) weak cation exchange (CM-650M) chromatography to reduce aggregates; and

(v) filtration to remove viruses.

The exact volumes, column sizes and operating parameters will depend, in part, on the scale of purification, as is well-known in the art. Specific volumes, column sizes and operating parameters useful for large-scale purifications are described in Section 7.4.

Crude daclizumab to be purified and optionally formulated via the above methods can be harvested from the cell culture using a variety of conventional means, e.g., microfiltration, centrifugation, and depth filtration directly from bioreactor. However, it has been discovered that crude daclizumab can be conveniently harvested by lowering the pH of the cell culture to approximately pH 5 at a temperature of less than 15° C. to flocculate the cells, which can be removed via centrifugation. In a specific embodiment, crude daclizumab is harvested by lowering the pH of the cell culture to approximately pH 5, chilling the culture to less than 15° C., for example 4° C., for 30-90 minutes, and centrifuging the resultant suspension to remove cells. This process is generally applicable to any cell culture that secretes recombinant proteins into the culture medium, and is not specific to cultures producing daclizumab or therapeutic antibodies. The pH of the culture can be adjusted using a variety of different acids, including weak or strong organic acids, or weak or strong inorganic acids. For daclizumab cultures, it has been discovered that citric acid works well. A concentrated citric acid solution, e.g., a 0.5 M-2 M solution, can be used for adjusting the pH of the culture prior to harvesting.

The purification of a DAC HYP is accomplished by use of three chromatography steps, virus inactivation, virus filtration and final ultra filtrations. Protein A affinity chromatography is the first step in the purification process, which clears the majority of process related impurities. To enable the reuse of protein A affinity column, it must be regenerated and sanitized. It has been discovered that aqueous NaOH solution is effective in accomplishing both column regeneration and sanitization. However, the use of NaOH solutions can degrade the protein A resin, increasing overall production costs. It has also been discovered that sanitizing protein A affinity chromatography resins with a solution containing NaOH and benzyl alcohol yields good results and significantly increases the number of purification cycles. The buffer generally comprises about 100 to 500 mM sodium citrate, about 10 to 30 mM NaOH and about 0.5 to 3% (v/v) of benzyl alcohol, and has a pH in the range of about pH 10 to 13. The buffer may also optionally include other components, such as, for example, salts and/or detergents. Both sodium citrate and benzyl alcohol are important for protecting protein A resin from being destroyed by NaOH and enhancing microbicidal activities. In specific embodiments, the Protein A sanitization buffer contains about 200 mM sodium citrate, about 20 mM NaOH, and about 1% (v/v) benzyl alcohol.

The sanitization buffer can be used to sanitize Protein A chromatography resin in a batch-wise process, where the resin is washed with excess (e.g., 1.5-2× volumes) of sanitization buffer followed by incubation for about 30-45 min. in excess (e.g., 1.5-2× volumes) sanitization buffer, followed by equilibration with equilibration buffer or storage buffer. The sanitization buffer can also be used to sanitize a prepared Protein A chromatography column by washing the column with excess (e.g., 1.5-2× column volumes) sanitization buffer at a suitable flow rate (e.g., ranging from about 110-190 cm/hr, or 135-165 cm/hr), holding the column under conditions of zero flow for about 30-40 min, and then washing the column with equilibration buffer or storage buffer. Suitable equilibration and storage buffers are described in Section 7.4.

DAC analogs expected to be useful in the methods described herein include IgG2, IgG3, and IgG4 anti-CD25 antibodies having specific CD25 binding properties similar to DAC, and to anti-CD25 antibodies suitable for administration to humans that have not been humanized, and fragments of DAC, including, for example, Fab fragments.

The daclizumab is generally administered in the form of a pharmaceutical composition formulated for subcutaneous administration. Formulations suitable for use in the methods disclosed herein are described in more detail, below.

The daclizumab may be administered as monotherapy, or it may be administered in conjunction with, or adjunctively to, other drugs useful to treat MS, including, for example, interferon beta, glatiramer acetate (e.g., Copaxone®, Teva Pharmaceutical Industries, Ltd., Israel), natalizumab, cladribine, corticosteroids, riluzole, azathioprine, cyclophosphamide, methotrexate, and mitoxantrone.

Dosages of these other drugs may be those used conventionally.

In the SELECT trial, the overall incidence of adverse events and treatment discontinuations were similar in all study arms. Serious infections (2% versus 0%), serious cutaneous events (1% versus 0%) and liver function test abnormalities greater than five times the upper limit of normal (4% versus <1%) occurred more frequently in DAC HYP-treated patients than in the placebo group. Although serious cutaneous events have been observed in other clinical trials using daclizumab, this is the first time that liver function abnormalities have been observed. Thus, in addition to monitoring patients treated with daclizumab for serious cutaneous events, the results from the SELECT trial suggest that patients should be monitored for liver enzyme abnormalities.

Liver enzyme abnormalities can be assessed using blood tests to detect the presence of certain liver enzymes (proteins) in the blood. Among the most sensitive and widely used liver enzymes are the aminotransferases. They include aspartate aminotransferase (AST or SGOT) and alanine aminotransferase (ALT or SGPT). In addition to AST and ALT, alkaline phosphatase, 5′ nucleotidase, and gamma-glutamyl transpeptidase (GGT) can also be used to measure liver function.

In some embodiments, AST/SGOT levels are monitored in patients treated with daclizumab. Normal range of values for AST/SGOT is from 5 to 40 units per liter of serum. In other embodiments, ALT/SGPT levels are monitored in patients being treated with daclizumab. Normal range of values for ALT/SGPT is from 7 to 56 units per liter of serum. In yet other embodiments, the levels of AST/SGOT and ALT/SGPT are monitored. The ranges of AST and ALT numbers may differ slightly depending on the technique and protocols used by different laboratories. Normal reference ranges are routinely provided by each laboratory and printed in the report.

Serum levels of AST/SGOT and/or SLT/SGPT should be determined prior to daclizumab treatment and at selected intervals thereafter. In some embodiments, serum levels of AST/SGOT and/or SLT/SGPT can be monitored prior to treatment with daclizumab and prior to each subsequent administration of daclizumab. For example, if daclizumab is to be administered monthly, serum levels of AST/SGOT and/or SLT/SGPT can be determined monthly. As will be appreciated by a person of skill in the art, the frequency of monitoring can be adjusted by the treating physician based on the clinical evaluation of the individual.

As will be appreciated by a person skilled in the art, elevated levels of AST/SGOT and ALT/SGPT do not automatically equate with liver disease. For example, elevations of these enzymes can occur with muscle damage. Accordingly, patients exhibiting serum levels of AST/SGOT and ALT/SGPT that exceed the upper limit of normal (ULN) by at least three fold following treatment with daclizumab should be further evaluated to determine if treatment with daclizumab should be suspended or discontinued. In other embodiments, patients having serum levels of AST/SGOT and ALT/SGPT that exceed the ULN by at least three, four, five, ten, twenty, thirty, forty, fifty, sixty, seventy, eighty, ninety, one hundred, two hundred, three hundred, four hundred, five hundred, six hundred, seven hundred, eight hundred, nine hundred, or a thousand fold should be further evaluated to determine if treatment with daclizumab should be suspended or discontinued. Additional tests that can be used to confirm if liver function is being negatively impacted, include the use of a coagulation panel (prothrombin time or PT, and international normalized ratio or INR), albumin level (hypoalbuminemia), bilirubin, and platelet count.

In some embodiments, treatment with daclizumab is temporarily suspended if a patient develops any of the following: ALT/SGPT or AST/SGOT >3 ULN, total bilirubin >2 ULN, and/or any other clinically significant hepatic test abnormality in the opinion of the treating physician. Treatment with daclizumab may be resumed when ALT/SGPT and AST/SGOT ≤2 ULN and total bilirubin is within the ULN. If treatment with daclizumab is resumed, the levels of ALT/SGPT and/or AST/SGOT should be re-evaluated between 2 and 4 weeks after receiving daclizumab.

7. EXEMPLARY EMBODIMENTS

Various aspects and features of the inventions described herein are described further by way of the exemplary embodiments, below. It will be appreciated that while the exemplary embodiments utilize specific cell culture media, cell culture conditions, column chromatography resins and equilibration, washing and elution buffers, routine changes can be made. Moreover, while the various cell culturing methods are exemplified with a specific producer strain (clone 7A11-5H7-14-43, also referred to as Daclizumab dWCB IP072911), it is expected that other DAC or DAC analog producer strains could be used with success, with or without routine optimization. Moreover, features that are described in association with a particular embodiment (whether in the Summary above or in the Exemplary Embodiments that follow) can be deviated from without substantially affecting the desirable properties of the methods and compositions of the disclosure, and moreover that different embodiments can be combined and used in various ways together unless they are clearly mutually exclusive. Accordingly, it is to be understood that the exemplary embodiments provided below are intended to be illustrative and not limiting, and should not be construed as limiting the claims that follow these embodiments.

The manufacturing method exemplified below was used to produce a DAC HYP drug substance at 150 mg/mL. For making a DAC HYP drug substance at 100 mg/mL, small process changes are introduced:

The cell culture used to produce a DAC HYP (see Section 7.3) at 100 mg/mL does not include an antifoam emulsion, whereas the cell culture DAC HYP at 150 mg/mL uses a low concentration Dow Corning Antifoam C in the 10,000 L bioreactor to minimize foaming. The CM-650M column (see Section 7.4.5) is sanitized with a buffer of 0.5 M NaOH, 0.5 M sodium sulfate when producing a DAC HYP formulation having a final antibody concentration of 100 mg/mL; the sodium sulfate is omitted from the sanitization buffer when producing a DAC HYP formulation having a final antibody concentration of 150 mg/mL. For making DAC HYP at 100 mg/mL, a one step ultrafiltration/diafiltration (UF/DF) is used at the end of the downstream process immediately prior to addition of polysorbate 80 and dilution of the drug substance to final volume (see Section 7.4.7), whereas for making the drug substance at a concentration to 150 mg/mL, a two step UF/DF is used. The examples below show comparative analyses among various lots of DAC HYP at 100 mg/mL and at 150 mg/mL. In several studies, batch of DAC HYP at 150 mg/mL were compared against a lot of DAC HYP 100 mg/mL manufactured at the 10,000 L scale, referred to below as Reference Standard lot RS0801.

7.1. DAC HYP Expression Construct

The hybridoma producing anti-Tac, a murine IgG_(2a) monoclonal antibody, was generated by fusing the murine myeloma cell line NS-1 with spleenocytes from a mouse immunized with a human T-cell line developed from a T-cell leukemia patient (Uchiyama et al., 1981, J. Immunol. 126(4):1393-7). Anti-Tac was selected for its reactivity with activated T-cells, but not with resting T-cells or B-cells. Anti-Tac was later shown to react with the alpha subunit of human IL-2 receptor (Leonard et al., 1982, Nature 300(5889):267-9).

The amino acid sequences for the light and heavy chain variable regions of the murine anti-Tac were determined from the respective cDNA (Queen et al., 1989, Proc. Nat'l Acad. Sci. USA 86(24):10029-33). The binding affinity of the mouse anti-Tac was retained in the humanized form as described in Queen et al. The complementarity determining regions (CDRs) of the murine anti-Tac were first grafted onto the acceptor framework of human antibody Eu. With the aid of a three-dimensional model, key mouse framework residues critical for the conformation of the CDRs and thus the binding affinity were identified and substituted for the human counterpart in the acceptor frameworks. In addition, atypical amino acids in the acceptor frameworks were replaced with the human consensus residues of the corresponding positions to eliminate potential immunogenicity.

DAC HYP V_(L) and V_(H) genes were constructed as mini-exons by annealing and extension of overlapping oligonucleotides as described in Queen et al. (1989). For expression of DAC HYP in the IgG₁ form, the resultant V_(L) and V_(H) genes were cloned into a single expression vector, as outlined in Cole et al. (1997, J. Immunol. 159(7):3613-21) and Kostelny et al. (2001, Int. J. Cancer 93(4):556-65), to construct pHAT.IgG1.rg.dE (see FIG. 3 and FIG. 4A). Plasmid pHAT.IgG1.rg.dE contains the genes for both the IgG₁ heavy and kappa light chains of daclizumab, each under control of the human cytomegalovirus (CMV) promoter. The plasmid contains the E. coli guanine phosphoribosyl transferase (gpt) gene as a selectable marker. The genetic components in pHAT.IgG1.rg.dE are described in Table 1 below.

TABLE 1 Genetic Components of pHAT.IgG1.rg.dE Nucleotide Restriction Number Sites in Vector Description Reference 0001-0623 EcoRI-XbaI CMV IE enhancer and promoter Boshart et al., 1985, Cell 41(2): 521-30 0624-1056 XbaI-XbaI DAC HYP V_(H) 1057-3852 XbaI-BamHI Human Cγ1 Ellison et al., 1982, Nucleic Acids Res. 10(13): 4071-79 3853-3981 BamHI-EcoRI Transcription termination site from human Ashfield et al., 1991, EMBO J. 10(13): 4197-207 complement gene C2 3982-4604 EcoRI-XbaI CMV IE enhancer and promoter Boshart et al., 1985, Cell 41(2): 521-30 4605-5001 XbaI-XbaI DAC HYP V_(L) 5002-6524 XbaI-BamHI Human CK Hieter et al., 1980, Cell 22(1 Pt 1): 197-207. 6525-6735 BamHI-HindIII SV40 enhancer and promoter Reddy et al., 1978, Science 200: 494-502 6736-7793 HindIII-Sau3AI E. coli gpt gene Richardson et al., 1983, Nucleic Acids Res. 11(24): 8809-16 7794-8403 Sau3AI-Sau3AI SV40 intron Reddy et al., 1978, Science 200: 494-502 8404-8639 Sau3AI-BamHI SV40 poly A Reddy et al., 1978, Science 200: 494-502 8640-10936 BamHI-EcoRI pBR322 region including amp gene Sutcliffe, 1979, Cold Spring Harb Symp Quant Biol. 43 Pt 1: 77-90

The dESV40 promoter spans positions of 6536-6735 of pHAT.IgG1.rg.dE (6536-6562 is 27 residues of 72 bp enhancer A; 6566-6629 are the three 21-bp repeats. 6536-6735 are the reverse complement of 5172-1 and 1-133 in GenBank: J02400.1 (Simian Virus 40 Complete Genome)). The nucleotide sequences of DAC HYP light and heavy chain genes in the expression vector were confirmed by DNA sequencing

7.2. DAC HYP Stable Cell Line

Mouse myeloma cell line NS0 was obtained from European Collection of Cell Cultures (ECACC catalog #85110503, Salisbury, Wiltshire, UK). A vial of these NS0 cells was thawed into DMEM supplemented with 10% FBS. Cells were maintained in a humidified incubator at 37° C. and 7.5% CO₂. The cells were subsequently cultured in basal medium SFM-3 supplemented with 1 mg/mL BSA. SFM-3 is a 1:1 mixture of DMEM and Ham's F-12 supplemented with 10 mg/mL insulin and 10 μg/mL transferrin. Over a period of approximately 3 months, the NS0 cells were adapted to SFM-3 without supplements, by gradually reducing the amount of FBS present in the culture medium until it was eliminated, and then finally removing BSA in a single step. The resulting host cell line was passaged 15-20 times in SFM-3 and a frozen bank was prepared.

The SFM-3 adapted cells were transfected with pHAT.IgG1.rg.dE (linearized with FspI enzyme (New England Biolabs, cat. no. R0135L, lot 43)) by electroporation. Briefly, 30-40 μg of pHAT.IgG1.rg.dE was added to 1×10⁷ exponentially growing adapted NS0 cells and pulsed twice at 1.5 kV, 25 F using a Gene Pulser instrument (BioRad, Richmond, Calif.). Following electroporation, cells were plated in DMEM±10% FBS in five 96-well plates at 20,000 cells/well, a density that favored a single colony per well after mycophenolic acid (“MPA”) selection. As described in Hartman et al., 2007, Biotech. & Bioeng 96(2):294-306, transfectants that had stably integrated the vector were selected in the presence of mycophenolic acid. Starting from an NS0 stable transfectant that produced a high level of DAC HYP, three successive rounds of subcloning were performed by either limited dilution cloning or fluorescence activated cell sorting (FACS) into PFBM-1 containing either 2.5% or 5% fetal bovine serum (FBS; HyClone, Logan, Utah). At each round of subcloning, one of the best producers was used for the next round of subcloning. Following the third round of subcloning, the final production cell line (7A11-5H7-14-43, also referred to as Daclizumab dWCB IP072911) was chosen. A seed bank of the final production cell line was then prepared by freezing 1×10⁷ cells per vial in 1 mL of 90% FBS/10% DMSO (Sigma, St. Louis, Mo.).

7.3. DAC HYP Recombinant Production

7.3.1. Cell Culture and Recovery

Cells are thawed from a single cell bank vial and expanded in progressively larger volumes within T-flasks, roller bottles, spinner flasks, and bioreactors until the production scale is achieved. Upon completion of the production culture, the cell culture fluid is clarified by centrifugation and depth filtration, and transferred to a harvest hold tank. The production culture duration is approximately 10 days.

Cell culture and recovery can be carried out in a variety of different cell culture facilities using standard equipment, as is known in the art. In another example, cells are thawed from a single cell bank vial and expanded in progressively larger volumes within shaker flasks and bioreactors until the production scale is achieved. Upon completion of the production culture, the cell culture fluid is clarified by centrifugation and depth filtration, and transferred to a harvest hold tank. The production culture duration is approximately 10 days.

7.3.1.1. Inoculum Preparation

Production batches are initiated by thawing a single cell bank vial. Cells are transferred to a T-flask containing a chemically-defined medium, Protein Free Basal Medium-2 (PFBM-2). Custom Powder for making PFBM-2 can be ordered from Invitrogen by requesting Hybridoma-SFM media powder prepared without NaCl, phenol red, transferrin, and insulin, including a quantity of EDTA iron (III) sodium salt that, when reconstituted, yields a concentration of 5 mg/L, and that has quantities of the remaining components adjusted such that, when reconstituted, their concentrations are the same as reconstituted Hybridoma-SFM. Prepared PFBM-2 medium contains the following components: 8 g/L Custom Powder; 2.45 g/L sodium bicarbonate; 3.15 g/L NaCl; and 16.5 g/L D-glucose monohydrate (15 g/L glucose).

The cells are expanded by serial passage into roller bottles or spinner flasks every two days thereafter. T-flasks, roller bottles, and spinner flasks are placed in an incubator operating under a temperature set point of 37° C. under an atmosphere of 7.5% CO₂ for T-flasks and roller bottles and 5% CO₂ for spinner flasks.

The spinner flasks are supplemented with 5% CO₂ either by overlay into the headspace or by sparge into the culture, depending on the cell culture volume, and impeller speed is controlled at constant revolutions per minute (RPM). The target seeding density at all inoculum expansion passages is approximately 2.5×10⁵ viable cells/mL.

Furthermore, inoculum preparation can be carried out according to methods known in the art, using a variety of standard culture vessels, volumes, and conditions. For example, production batches can be initiated by thawing a single cell bank vial. Cells can be transferred to a shaker flask containing a chemically-defined medium, Protein Free Basal Medium-2 (PFBM-2). Custom Powder for making PFBM-2 can be ordered from Invitrogen by requesting Hybridoma-SFM media powder prepared without NaCl, phenol red, transferrin, and insulin, including a quantity of EDTA iron (III) sodium salt that, when reconstituted, yields a concentration of 5 mg/L, and that has quantities of the remaining components adjusted such that, when reconstituted, their concentrations are the same as reconstituted Hybridoma-SFM. Prepared PFBM-2 medium contains the following components: 8 g/L Custom Powder; 2.45 g/L sodium bicarbonate; 3.15 g/L NaCl; and 16.5 g/L D-glucose monohydrate (15 g/L glucose). Optionally, at the bioreactor stage, cupric sulfate heptahydrate can be added, e.g., at a concentration of 0.04 mg/L.

The cells are expanded by serial passage into shaker flasks every two days thereafter. Shaker flasks are placed in an incubator operating under a temperature set point of 37° C. under an atmosphere of 7.5% CO₂.

The shaker flasks are agitated at constant revolutions per minute (RPM) on a shaker platform in the incubators. The target seeding density at all inoculum expansion passages is approximately 2.2-2.5×10⁵ viable cells/mL.

Approximately 14 days following cell bank thaw, when a sufficient number of viable cells have been produced, the first of several, typically three or four, stainless steel stirred-tank seed bioreactors is inoculated. Prior to use, the seed bioreactors are cleaned-in-place, steamed-in-place, and loaded with the appropriate volume of PFBM-2 culture medium. The pH and dissolved oxygen probes are calibrated prior to the bioreactor being steamed-in-place. The first seed bioreactor is inoculated with a sufficient number of cells to target an initial cell density of 2.0-2.5×10⁵ viable cells/mL. Sequential transfer to the larger volume (typically, 100 L to 300 L and then to the 1,000 L seed bioreactors, or 60 L to 235 L, 950 L, and 3750 L seed bioreactors) is performed following approximately two days of growth in each reactor and target initial cell densities of 2.0-2.5×10⁵ viable cells/mL. Culture pH is maintained by addition of either CO₂ gas or 1 M sodium carbonate (Na₂CO₃) via automatic control. The target operating conditions of the seed and production bioreactors include a temperature set point of 37° C., pH 7.0 and 30% dissolved oxygen (as a percentage of air saturation). The 100 L, 300 L and 1,000 L bioreactors are agitated at 100 rpm, 80 rpm and 70 rpm, respectively. In some instances, the target operating conditions of the seed and production bioreactors include a temperature set point of 37° C., a pH of 7.0 with CO₂ sparge and base addition control and 30% dissolved oxygen (as a percentage of air saturation). The larger volume bioreactors can be agitated at speeds of 100 rpm, 80 rpm, 70 rpm, or 40 rpm.

7.3.2. Cell Culture Production Bioreactor

After approximately 2 days in the 1,000 L seed bioreactor, the inoculum is transferred into a stainless steel stirred-tank production bioreactor. The production bioreactor has a working volume of approximately 10,000 L. Prior to use, the bioreactor is cleaned-in-place, steamed-in-place, and loaded with approximately 4,000 L of PFBM-2 medium. The pH and dissolved oxygen probes are calibrated prior to the bioreactor being steamed-in-place.

In another example, the inoculum is grown in a 3750 L seed bioreactor before transfer to a stainless steel stirred-tank production bioreactor with a working volume of approximately 15,000 L, which is cleaned-in-place, steamed-in-place, and loaded with approximately 4,000-7,000 L of PFBM-2 medium prior to use.

The target seeding density of the production bioreactor is in the range of 2.0-2.5×10⁵ viable cells/mL. A chemically-defined Protein Free Feed Medium concentrate (PFFM-3) (a chemically-defined concentrated feed medium made by reconstituting PFFM3 subcomponents 1 and 2, L-glutamine, D-glucose, sodium phosphate dibasic heptahydrate, L-tyrosine, folic acid, hydrochloric acid, and sodium hydroxide) is added during culture. PFFM3 contains the components shown in Table 4:

TABLE 4 PFFM3 Medium Components Component Concentration PFFM3 Subcomponent 1 (amino acids) 20.4 g/L prepared PFFM3 Subcomponent 2 4.93 g/L prepared (vitamins and trace elements) L-Glutamine 11.0 g/L prepared D-Glucose 28.0 g/L prepared L-Tyrosine, disodium salt 1.32 g/L prepared Folic Acid 0.083 g/L prepared  Na₂HPO₄•7H₂0 1.74 g/L prepared Sodium Hydroxide Varies, pH control Glacial Hydrochloric Acid Varies, pH control WFI water

PFFM3 Subcomponent 1 contains the components shown in Table 5 below:

TABLE 5 PFFM3, Subcomponent 1 MW Conc. Conc. Medium Components (g/mole) (mg/L) (mM) L-Arginine HCl 211. 1,900 9.00E+00 L-Asparagine Anhydrous 132.1 1,320 9.99E+00 L-Aspartic Acid 133.1 119 8.94E−01 L-Cysteine HCl•H₂O 176.0 2,030 1.15E+01 L-Glutamic Acid 147.1 510 3.47E+00 Glycine 75.1 157 2.09E+00 L-Histidine HCl•H₂O 210.0 864 4.11E+00 L-Isoleucine 131.2 1,440 1.10E+01 L-Leucine 131.2 3,130 2.39E+01 L-Lysine HCl 183.0 2,160 1.18E+01 L-Methionine 149.2 1,260 8.45E+00 L-Phenylalanine 165.2 918 5.56E+00 L-Proline 115.1 806 7.00E+00 L-Serine 105.1 709 6.75E+00 L-Threonine 119.1 1,220 1.02E+01 L-Tryptophan 204.2 408 2.00E+00 L-Valine 117.1 1,450 1.24E+01

PFFM3 Subcomponent 2 contains components shown in Table 6 below:

TABLE 6 PFFM3, Subcomponent 2 MW Conc. Conc. Medium Components g/mole) (mg/L) (mM) Vitamin B-12 1,355.0 10.72 7.91E−03 Biotin 244.0 0.156 6.39E−04 Choline Chloride 140.0 140 1.00E+00 I-Inositol 180.0 197 1.09E+00 Niacinamide 122.0 31.5 2.58E−01 Calcium Pantothenate 477.0 103.1 2.16E−01 Pyridoxine Hydrochloride 206.0 0.484 2.35E−03 Thiamine Hydrochloride 337.0 99.8 2.96E−01 Putrescine 2HCl 161.1 6.66 4.13E−02 DL-Lipoic thioctic acid 206.0 4.84 2.35E−02 Sodium Pyravate 110.0 1,716 1.56E+01 Ethanolamine HCl 97.54 76.1 7.80E−01 β-Mercaptoethanol 78.13 60.9 7.80E−01 Linoleic Acid 280.48 0.655 2.34E−03 Pluronic F-68 8,350.0 780 9.34E−02 Potassium Chloride 74.55 432 5.79E+00 Riboflavin 376.0 3.42 9.09E−03 Magnesium Chloride Anhyd. 95.21 446 4.69E+00 Magnesium Sulfate Anhyd. 120.4 762 6.33E+00 Sodium Selenite 172.9 0.140 8.12E−04 Cupric Sulfate•5H₂O 249.7 0.1069 4.28E−04 Ferrous Sulfate•7H₂O 278.0 6.51 2.34E−02 Potassium Nitrate 101.1 0.593 5.86E−03 Zinc Sulfate•7H₂O 287.5 15.0 5.23E−02 Manganese Sulfate, 169.01 0.00264 1.56E−05 Monohydrate Nickelous Chloride, 237.7 0.00186 7.81E−06 6-Hydrate Stannous Chloride 2H₂O 225.63 0.001130 5.01E−06 Ammonium Molybdate 4H₂O 1,235.86 0.00193 1.57E−06 Ammonium meta-Vanadate 116.98 0.00913 7.80E−05 Sodium meta-Silicate 9H₂O 284.2 2.22 7.79E−03 EDTA, Iron(III), Sodium Salt 367.05 31.2 8.50E−02

The timing and amount of addition of PFFM-3 to the culture occurs as shown in Table 7 below:

TABLE 7 Exemplary DAC HYP Bioreactor Feed Schedule Day PFFM-3 Amount (% of initial mass) 0 0 1 0 2 4-4.14 3 7.8-8.08  4 7.8-8.08  5 7.8-8.08  6 11-11.38 7 13-13.46 8 15-15.52 9 15-15.52 10 0

Culture pH is maintained at approximately pH 7.0, preferably between pH 7.0 and pH 7.1, by automatic control of CO₂ gas and 1 M sodium carbonate (Na₂CO₃) addition. Dissolved oxygen content is allowed to drop to approximately 30% of air saturation. An oxygen/air mixture is sparged into the culture to achieve a constant total gas flow rate and dissolved oxygen is controlled by adjusting the ratio of air to oxygen gases as needed and by increasing agitation speed after reaching a maximum oxygen to air ratio. In another example, agitation is adjusted to maintain a constant power/volume ratio. A simethicone-based antifoam emulsion is added to the bioreactor on an as needed basis based on foam level. Samples are taken periodically to test for cell density, cell viability, product concentration, glucose and lactate concentrations, dissolved O₂, dissolved CO₂, pH, and osmolality. The bioreactor culture is harvested approximately 10 days post-inoculation. Prior to harvest, the bioreactor contents are sampled as unprocessed bulk.

7.3.3. Harvest and Cell Removal

Just prior to harvest, the production bioreactor is first chilled to <15° C., then adjusted to a pH of 5.0±0.1 using 0.5 M or 1 M or 2 M citric acid, and held for a period of approximately 30-90 or 45-60 minutes to flocculate the cells and cell debris prior to transfer to the harvest vessel. The pH-adjusted harvest is then clarified by continuous centrifugation operated under predefined parameters for bowl speed and flow rate as defined in batch record documentation.

The centrate is filtered through a depth filter followed by a 0.22 μm membrane filter and collected in a pre-sterilized tank. The cell-free harvest is adjusted to an approximate pH of 6.4 using a 1-2 M Tris solution and stored at 2-8° C. for further processing. In some instances, this pH adjustment occurs within 12 hours of the original bioreactor pH adjustment to pH 5.0.

7.4. DAC HYP Purification

7.4.1. Overview

The DAC HYP purification and formulation process was designed to improve efficiency relative to the ZENAPAX production process and to ensure consistent clearance of product- and process-related impurities. The following subsections describe the purification process. The purification is based on three chromatography techniques (Protein A affinity chromatography, Q Sepharose anion exchange chromatography, and CM-650(M) cation exchange chromatography) in combination with low pH viral inactivation, viral filtration, ultrafiltration/diafiltration, and formulation steps. All of the steps take place in enclosed equipment. An outline of the purification process for DAC HYP is presented in FIG. 5 and described below.

7.4.2. Protein A Chromatography

The Protein A affinity chromatography step is the first purification step in the sequence of downstream operations. This step occurs in one or more cycles depending on the size of the column, typically two or three cycles for the column described in Table 8A (i.e., the cell-free harvest is portioned into two aliquots, and then each aliquot is loaded and eluted separately on the Protein A column). Recombinant Protein A affinity chromatography resin specifically binds IgG, separating antibody from other components of the cell culture harvest.

Following equilibration of the Protein A column with an equilibration buffer, the neutralized, cell-free harvest is passed through the column in order to bind the antibody to the column resin. The equilibration buffer is 20 mM sodium citrate, 150 mM sodium chloride, pH 7.0. The column is loaded to a capacity of no greater than 35 grams antibody (protein) per liter of the packed resin. Following loading, the column is washed with the equilibration buffer to remove the unbound and loosely bound impurities from the resin, as well as a pre-elution wash with a citrate buffer to adjust the citrate and sodium chloride concentration of the column. The citrate buffer is 10 mM sodium citrate pH 7.0. The bound antibody is then eluted from the column with a step change in pH using an elution buffer of 10 mM sodium citrate at pH 3.5. A summary of the Protein A chromatography conditions is set forth in Table 8A:

TABLE 8A Exemplary DAC HYP Protein A chromatography parameters PARAMETER SET POINT Resin MabSelect Column bed height, cm 10-25, typically 14 Column diameter, cm 1-120, depending on scale Operation temperature 5° C. Equilibration/wash buffer 20 mM NaCitrate, 150 mM NaCl, pH 7.0 Equilibration volume, CV 5-7 Equilibration flow rate, cm/hr 150-300 Load flow rate, cm/hr 150-300 Load capacity, grams IgG/L resin <35  Wash flow rate, cm/hr 150-300 Wash volume, CV 7 Pre-Elution conditioning Follow 7 CV wash with 2 CV of 10 mM NaCitrate, pH 7.0 and flow rate of 150-300 cm/hr Elution buffer 10 mM NaCitrate, pH 3.5 Elution flow rate, cm/hr 150-300 Collection criteria 250 mAU - 250 mAU (UV detector path-length: 5.0 mm) Elution buffer volume post 2 elution, CV Flow direction, equilibration, Down sanitization and storage Equilibration 20 mM NaCitrate, 150 mM NaCl, pH 7.0 Equilibration volume, CV 2-3 Equilibration flow rate, cm/hr 150-300 Sanitization buffer 200 mM NaCitrate, 20 mM NaOH, 1% Benzyl Alcohol Sanitization flow rate, cm/hr 150 cm/hr for 1.8 CV, then hold for 30 mins. Equilibration for next cycle 5-7 CV Storage buffer 200 mM sodium citrate, 1% benzyl alcohol, pH 7.0 Storage flow rate, cm/hr 150-300 Storage buffer volume, CV 4 CV Column Storage temp, ° C. 5° C.

As the product elutes off the column, the absorbance of the effluent at a wavelength of 280 nm is monitored and used to guide the collection of the product fraction (see FIG. 6).

The use of a sanitization buffer containing sodium hydroxide and benzyl alcohol advantageously kills a wide range of microbial organisms while minimally affecting the quality of the protein A resin. To illustrate this, various sanitization solutions were spiked with various microorganisms and incubated over a period of time. At different intervals of incubation time, portions of the spiked sanitization solutions were neutralized and the microorganism titers were measured and compared to control. The microbicidal activities are expressed in the log reduction of the microorganisms over a period of time. Table 8B shows the reduction of microorganism titers as function of contact time with sanitization buffer 20 mM sodium hydroxide, 200 mM sodium citrate and 1% benzyl alcohol:

TABLE 8B Reduction of microorganism titers as function of contact time with sanitization buffer 20 mM sodium hydroxide, 200 mM sodium citrate and 1% benzyl alcohol LRV@ LRV@ LRV@ LRV@ LRV@ Organism 0 min 15 min 30 min 60 min 120 min E. coli (Gram negative) >5.7 >5.7 >5.7 >5.7 >5.7 S. aureus 1.1 >5.1 >5.1 >5.1 >5.1 (Gram positive) B. subtilis 2.8 2.7 3.2 3.1 3.6 (spore forming) (Gram negative) P. aeruginosa >5.0 >5.0 >5.0 >5.0 >5.0 (Gram negative) C. albicans (yeast) 4.2 >5.5 >5.5 >5.5 >5.5 A. niger (fungus) −0.2 0.4 0.5 0.8 1.4

Table 8 C shows the reduction of microorganisms by different sanitization solutions:

TABLE 8C Log₁₀ Reduction of microorganisms by different solutions A B C D E. coli >3.6 >5.7 >4.0 S. aureus >3.6 6.0 0.5 >4.0 Micrococcus lylae 3.3 Bacillus sp. (spore forming) B. subtilis −0.3 3.1 0.2 0.12 Paenibacillus −0.01 −0.03 glucanolyticus B. cereus 5.0 Pseudomonas sp. P. aeruginosa >3.6 >4.6 Stenotrophomonas 6.0 altophilia Candida albicans (Yeast) 3.1 >5.5 0 >4.8 Aspergillus niger 0.01 0.8 0 >4.7 A = 50 mM NaOH, 0.5M NaCl (60 min) B = 20 mM NaOH, 200 mM sodium citrate, 1% benzyl alcohol (60 min) C = 200 mM sodium citrate, 0.5% benzyl alcohol (48 hrs) D = 2% benzyl alcohol (24 hrs)

The forgoing data shows that sanitization solutions containing benzyl alcohol and sodium hydroxide are very effective in killing a wide variety of microorganisms, including gram negative and gram positive bacteria, spore forming bacteria, yeast and fungus. After 30 minutes of typical sanitization, more than 5 log₁₀ reductions were observed on E. coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans. Although the killing of fungus (A. niger) took longer, it is rare to have fungus infection in the cell culture fluids. The most common microorganisms isolated in biotech facility are Bacillus, Pseudomonas and Staphylococcus. These are effectively killed by the sanitization solution after 30 minutes of contact time. In comparison, sodium hydroxide or benzyl alcohol alone are not effective in killing all the microorganisms. Moreover, the sodium hydroxide sanitization solution does not kill spore forming Bacillus.

7.4.3. Low pH Hold for Viral Inactivation

This step is designed to inactivate low pH-sensitive endogenous virus-like particles and viruses. The Protein A eluate from each Protein A cycle is eluted into a collection tank, where 0.5 M HCl is added until a pH 3.5±0.1 is reached. The product is transferred to a hold tank where the pH is verified by another pH meter. The low pH hold step is tightly controlled at pH 3.5±0.1 or 0.2 (e.g., pH 3.35-3.64) for 30-120 minutes, or 30-240 minutes. After 30-120 minutes hold, the viral inactivated eluate is neutralized to a pH of 7.8±0.1 or +0.3 (e.g., pH 8.05-8.34) using 1 M Tris base, and then transferred through a 0.22 μm filter into a product pool tank. A summary of the low pH viral inactivation conditions is set forth in the Table 9:

TABLE 9 DAC HYP low pH viral inactivation parameters PARAMETER SET POINT Inactivation pH 3.5 Dilution of eluate Dilute to <13 mg/mL and above tank sample point Dilution buffer 17 mM NaCl or elution buffer pH adjustment buffer 0.5M HCl Inactivation time 120 min at 5° C. or 30 min at ambient temp. Neutralization Buffer 1M Tris Post Neutralization pH Target 7.8 measured at 25° C.* *Alternatively, the post-neutralization pH target can be 8.2 at 25° C.

7.4.4. Q Sepharose Anion Exchange Flow Through Chromatography

The Q Sepharose anion exchange chromatography step is used to reduce product- and process-related impurities (e.g., nucleic acids, host cell proteins, product aggregates, leached Protein A ligand, etc.) and to provide additional viral clearance capacity to the purification process. The conductivity and pH of the load are chosen in a manner such that the antibody flows through the column and negatively-charged impurities, such as host cell proteins and cellular DNA, bind to the positively-charged resin.

The anion exchange column is equilibrated with an equilibration buffer of 20 mM Tris, 20 mM sodium chloride, pH 7.8. The pH-adjusted product from the low pH hold step is loaded onto the column to a capacity of no greater than 60 grams of antibody (protein), or no greater than 30-60 grams of antibody (protein), per liter of packed resin. Following the completion of loading, unbound antibody and impurities are removed from the column with the equilibration buffer.

Collection of the product is guided by monitoring the absorbance of the effluent at a wavelength of 280 nm (see FIG. 7).

The sanitization flow rate is 100 cm/hr and the hold time is 60 min.

A summary of the Q-sepharose chromatography conditions is set forth in Table 10:

TABLE 10 DAC HYP Q-Sepharose chromatography parameters PARAMETER SET POINT Resin Q Sepharose FF Column bed height (cm) 10-30, typically 19 Column diameter (cm) 1-120, depending on scale Operation temperature 5° C.-25° C. Flow direction for Down equilibration/load/ wash/regeneration/sanitization Equilibration/wash buffer 20 mM Tris, 20 mM NaCl, pH 7.8 Equilibration volume (CV)  8 Flow rate (cm/hr) 100 Load capacity (g/L) <60 Wash flow rate (cm/hr) 100 Collection criteria 0.25 AU - 0.25 AU (UV detector path-length: 5.0 mm) Regeneration/sanitization buffer 0.5M NaOH, 1M NaCl Regeneration/sanitization volume (CV)    1.8 Regeneration/sanitization hold buffer 0.5M NaOH, 1M NaCl Regeneration/sanitization time (min)  60 Storage buffer 12 mM NaOH Storage buffer volume (CV)  4 Column storage temp. (° C.) 5° C.

For some uses, the storage buffer volume set point is 3 and the column storage temperature is set at 5°-25° C.

7.4.5. CM-650(M) Cation Exchange Chromatography

This chromatography step is the last step used in the process to reduce trace levels of process- and product-related impurities. In addition to reducing aggregates and cleavage fragments of the antibody, this step also reduces process-related impurities such as host cell nucleic acids and proteins, and leached Protein A.

The column is equilibrated with an equilibration buffer of 20 mM sodium citrate, pH 4.5. The anion exchange eluate pool is adjusted to a pH of 4.5±0.1 or 0.2 (e.g., 4.35-4.64) using 0.5 M citric acid and loaded onto the column to a target loading capacity of no greater than 25 or 30 grams of antibody (protein) per liter of packed resin. Following the binding step, the column is washed with the equilibration buffer to remove any unbound, or loosely-bound, impurities from the resin. The bound antibody is then eluted from the column in a step elution mode with an elution buffer of 20 mM sodium citrate, 75 mM sodium sulfate, pH 4.5. Peak collection is guided by monitoring the absorbance of the effluent at a wavelength of 280 nm (see FIG. 8).

A summary of the CM-650(M) chromatography conditions is set forth in Table 11:

TABLE 11 DAC HYP CM-650(M) chromatography parameters PARAMETER SET POINT Resin CM650 (M) Column bed height (cm) 10-30, typically 18 Column diameter (cm) 1-140, depending on scale Operation temperature 20° C. Equilibration/wash buffer 20 mM NaCitrate, pH 4.5 Equilibration volume (CV) 5 Flow rate (cm/hr) 100  Load preparation Adjust pH of load to 4.5 with 0.5M citric acid Load capacity (g/L) ≤30  Wash volume (CV) 3 CV with equilibration buffer Elution buffer 20 mM NaCitrate, 75 mM Na₂SO₄, pH 4.5 Cation exchange product pool 1.25 AU up - 1.25 AU collection criteria (Based on UV detector path-length of 5.0 mm) IgG concentration diluted to   8.5 Dilution Buffer* 20 mM NaCitrate, pH 4.5 Regeneration/sanitization buffer 0.5M NaOH Regeneration/sanitization volume (CV)   1.8 Regeneration/sanitization hold time (min) 60 Storage buffer 12 mM NaOH Storage buffer volume (CV)  4 *Optionally, dilution buffer is not used.

7.4.6. Nanofiltration

The purpose of the nanofiltration step is to provide additional viral clearance capacity to the purification process. The removal of viruses and virus-like particles at this step occurs through a size-exclusion mechanism. The pores of the filter are designed such that the antibody passes through the filter whereas the virus-like particles and viruses are retained on the upstream side of the filter.

The cation exchange eluate that has been filtered through a 0.22 μm or 0.1 μm filter is passed through a small virus-retaining nanofilter, followed by a filter flush with DAC HYP formulation buffer without polysorbate 80 (40 mM succinate, 100 mM sodium chloride, pH 6.0). The buffer flush step is applied to recover antibody that remains in the line and filter housing.

A summary of the nanofiltration parameters is set forth in Table 12:

TABLE 12 Nanofiltration parameters PARAMETER SET POINT Pre-filter Ultipor Nylon filters Virus Filter V-Pro Magnus 2.2 or NFP Pressure, psig 20-30, typically 25 WFI Flush 100 L/m²* Equilibration buffer and wash 40 mM sodium succinate, buffer post load 100 mM NaCl, pH 6.0 Equilibration (L) 33 L/m²** Wash buffer volume (L) 33 L/m²† Process capacity Up to 371 L/m² *or >50 L/m² **or >50 L/m² †or approximately 50 L/m²

7.4.7. Ultrafiltration/Diafiltration (UF/DF)

This process step is designed to concentrate the product and exchange the buffer in the product to the DAC HYP formulation buffer without polysorbate 80. It is operated in a tangential flow mode using a 30 kDa nominal molecular weight cutoff membrane. Two ultrafiltration/diafiltration stages are used to produce 150 mg/mL formulation due to the expected product volume at the final concentration and relative hold-up volume of each UF system.

The first stage is processed using a large UF system (see FIG. 9). The nanofiltration filtrate is first concentrated to approximately 30 mg/mL and then diafiltered into exchange buffer (formulation buffer without polysorbate 80). The diafiltered antibody solution is further concentrated to approximately 100 mg/mL, then recovered at a concentration of approximately 55 mg/mL from the UF/DF system and transferred through a 0.22 m filter. The diafiltered antibody solution can also be recovered at a concentration of approximately 20-60 mg/mL. A summary of the parameters of the first stage is set forth in Table 13:

TABLE 13 UF/DF stage 1 parameters UF/DF Membrane: Millipore (Pellicon 2 or Pellicon 3, 30 KD MWCO) PARAMETER SET POINT Membrane Loading capacity (g/m2) ≤300  Inlet pressure (psig) 25 Outlet pressure (psig) 15 Permeate pressure (psig) Unrestricted/restricted Equilibration/Diafiltration buffer 40 mM sodium succinate, 100 mM NaCl pH 6.0 Concentration during diafiltration (g/L) 30 Diafiltration volume (exchange volumes) 10 Final concentration before product ~100  recovery (g/L) Membrane Reuse Yes Filter Flush 40 mM succinate, 100 mM NaCl pH 6.0 Sanitization Buffer (pre-use) 0.5M NaOH* Sanitization Time (mins) (pre-use)  60** Sanitization Temperature (° C.) (pre-use) Room Temperature Sanitization Flush WFI Sanitization Solution (post-use) 0.1M NaOH Sanitization Time (mins) (post-use) 60 Sanitization Temperature (° C.) (post-use) Room Temperature Storage Buffer 0.1M NaOH Product Concentration (g/L)  70† Product Storage (° C.) 2-20, typically 20 *or 0.1M NaOH **or 2 times 40 minutes †or ranging from 20-70

The second stage is processed using a smaller UF system, but with the same 30 kDa cutoff membrane. The DAC HYP (typically 55 mg/mL) solution is concentrated to approximately 180 mg/mL, recovered from the UF system, and then transferred through a 0.22 m filter. The UF system is rinsed with formulation buffer without polysorbate 80 and transferred through the 0.22 μm filter obtaining the purified drug substance at approximately 170 mg/mL or approximately 150-170 mg/mL. A summary of the parameters of the second stage is set forth in Table 14:

TABLE 14 DAC HYP UF/DF stage 2 parameters UF/DF Membrane: Millipore (Pellicon 2 or Pellicon 3, 30 KD MWCO) PARAMETER SET POINT Membrane Surface Area (m²) 0.005 to 20.5, depending on scale Inlet pressure (psig)  20 Outlet pressure (psig) Unrestricted Permeate pressure (psig) Unrestricted Equilibration buffer 40 mM sodium succinate, 100 mM NaCl pH 6.0 Final concentration before product 180 recovery (g/L) Recirculation/Flush system volume (L) 2 mL to 5 L, depending on scale Recirculation/flush system buffer 40 mM sodium succinate, 100 mM NaCl, pH 6.0 Sanitization Flush WFI Sanitization Solution (post-use) 0.1M NaOH Sanitization Time (mins) (post-use)  60* Storage Buffer 0.1M NaOH 0.2 μm multimedia filter for 1 × 30 inch filters post final concentration **or 2 times 40 minutes

7.5. DAC HYP Formulation

The final process step is the dilution of the purified drug substance to a final target concentration of 150 or 100 mg/L+10%, i.e., a final target concentration of 150±15 mg/mL (in the case of the 150 mg/mL formulation) or 100±10 mg/mL (in the case of the 100 mg/mL formulation) in buffer containing an appropriate concentration of polysorbate 80. The formulation is performed in stages.

For example, first, the formulation buffer without polysorbate 80 (40 mM sodium succinate, 100 mM sodium chloride, pH 6.0) is added to the purified drug substance to reach the 90% target volume of formulated drug substance. Then, a calculated amount of polysorbate 80 dilution buffer (40 mM succinate, 100 mM sodium chloride, 1% polysorbate 80, pH 6.0) is added to reach the target concentration of 0.03% (w/v) polysorbate 80 in the final formulation. Finally, the product volume is adjusted, using the formulation buffer (made of succinate and succinic acid for the 150 mg/mL formulation, and succinate and HCl for the 100 mg/mL formulation) without polysorbate 80, to achieve a final antibody concentration of 150±15 mg/mL (preferably 150±8 mg/mL). The 100 mg/mL drug product is formulated in a similar manner to a final concentration 100±10 mg/mL (preferably 100±5 mg/mL).

The formulated drug substance is filtered through a 0.22 μm filter into a BioProcess Container™ (BPC®) bag (or equivalent) which is placed inside a semi-rigid cylindrical support. The support encloses the BPC with a lid and provides a protective barrier between the flexible bag and the external environment. The formulated drug substance is stored at 2-8° C. in an access-controlled cooler for drug product fill/finish operations.

A summary of the formulation conditions is set forth in Table 15:

TABLE 15 150 mg/ml DAC HYP formulation conditions 0.2 μm filter for post formulation pool PARAMETER SET POINT Dilution buffer 40 mM sodium succinate, 100 mM NaCl, pH 6.0 Polysorbate 80 Stock Solution 40 mM sodium succinate, 100 mM NaCl, pH 6.0, 1% (w/v) polysorbate 80 Final Polysorbate 80 concentration (w/v) % 0.03 Final Daclizumab concentration (mg/mL) 100 or 150 Product Storage (° C.) 2-8, commonly 5

1 mL of the drug product is filled into vials or a syringe. A summary of the components of the finished 150 mg/mL and 100 mg/mL products have the components shown in Table 16 (all quantities are nominal values):

TABLE 16 Composition of the 150 mg/mL and 100 mg/mL DAC HYP Drug Product Formulations Drug product: Drug product: Ingredient 150 mg/mL 100 mg/mL DAC HYP 150 mg  100 mg  Sodium Succinate 5.9 mg 6.5 mg Succinic Acid 0.4 mg Not applicable Sodium Chloride 5.8 mg 5.8 mg Polysorbate 80 0.3 mg 0.3 mg Hydrochloric Acid Not applicable As needed for adjustment to pH 6.0 Sodium Hydroxide As needed for As needed for adjustment to pH 6.0 adjustment to pH 6.0 Water for Injection As needed for As needed for final volume final volume

7.6. Characterization of DAC HYP Drug Product

DAC HYP is glycosylated at amino acid 296 of both heavy chain subunits, with the major oligosaccharide form existing as a core fucosylated biantennary structure lacking terminal galactose.

The N-terminus of DAC HYP heavy chain exists as three major forms: 1) N-terminal glutamine (predicted from the DNA sequence), 2) N-terminal pyroglutamate (from the cyclization of N-terminal glutamine), and 3) N-terminal valine, histidine and serine residues in addition to the predicted N-terminal glutamine residue (results from incomplete cleavage of the signal peptide).

The C-terminus of DAC HYP heavy chain exists with and without the C-terminal lysine residue. The major form lacks the C-terminal lysine residue, resulting in a C-terminal glycine.

DAC HYP has a calculated molecular weight of 144 kDa based on the primary amino acid composition defined by the nucleotide sequence. The corresponding molecular weight of the reduced heavy chain is 48.9 kDa and the reduced light chain is 23.2 kDa; these weights do not account for carbohydrate content or post-translational modifications.

DAC HYP binding is highly specific for CD25, which is expressed on activated but not on resting T and B lymphocytes. DAC HYP binding to CD25 on these activated cells blocks the binding of IL-2 to CD25 and subsequent formation of the high affinity IL-2 receptor complex. Consequently, IL-2-induced proliferation of the activated cells is blocked. The observed and potential therapeutic efficacy of DAC HYP is believed to rest in large part on its inhibitory effect on the proliferation of activated autoreactive T-cells. However, DAC HYP might also exert a therapeutic effect through its blocking effect on other CD25-bearing cell types such as eosinophils.

To confirm that high concentration 150 mg/ml DAC HYP formulations were suitable for clinical investigations, a comprehensive physicochemical and biological evaluation was performed to characterize and compare two batches of DAC HYP 150 mg/mL drug substance, referred to herein as Batch 1 and Batch 2 (or Batch 150-1 or Batch 150-2, respectively), to Reference Standard lot RS0801, which is from a lot of DAC HYP 100 mg/mL manufactured at the 10,000 L scale.

The results demonstrate that the DAC HYP drug product 150 mg/mL lots are of high purity, are comparable to the 100 mg/mL lots, and are suitable for use in clinical studies. A summary of these characteristics is shown in Table 17:

TABLE 17 Purity characteristics of DAC HYP (all concentrations are nominal) 150 mg/mL lots 100 mg/mL lots Useful Criteria Category Test (2 batches) (24 batches) (for 150 mg/ml) Quality Color, appearance Colorless, clear to Colorless, clear to Colorless, clear to and clarity slightly opalescent slightly opalescent slightly opalescent liquid, essentially free liquid; no visible liquid, essentially free of visible particles particles of visible particles pH determination 6.0 at 25° C. 6.0-6.1 at 25° C. 5.8-6.2 at 25° C. Product 143-148 mg/mL 96.0-103 mg/mL 135-165 mg/mL concentration by UV spectroscopy Cation Exchange Chromatogram Not available Chromatogram Chromatography consistent with consistent with reference reference Identity Anti-idiotype Identifies as Identifies as Identifies as ELISA daclizumab daclizumab daclizumab Cation exchange Not applicable Chromatogram Not applicable chromatography consistent with reference Purity Size exclusion 99.3% to 99.4% 99.2% to 99.6% ≥95% main peak chromatography main peak main peak ≤3% aggregate 0.6% to 0.7% 0.4% to 0.8% aggregate aggregate SDS-PAGE 97.3% purity 95.8%-97.0% purity ≥93% purity (colloidal blue stain) reduced SDS-PAGE (silver Sample staining Sample staining Sample staining stain) reduced and consistent with consistent with consistent with non-reduced reference reference reference DNA content <0.1 pg/mg <0.01-<0.06 pg/mg ≤0.25 pg/mg Residual protein A <1 ppm-1 ppm <1 ppm ≤ 30 ppm (based on weight of protein A relative to weight of antibody) Host cell protein <1 ppm <1 ppm-5 ppm ≤50 ppm (weight basis) (weight basis) (based on weight of host cell protein relative to weight of antibody) Potency Binding potency 86%-105% relative 78%-129% relative 70%-130% relative potency potency potency Functional 95%-101% relative 73%-121% relative 70%-130% relative potency potency potency potency Safety Bacterial <0.01 EU/mg <0.01 EU/mg ≤0.751 EU/mg endotoxins Bioburden 0 CFU/100 mL 0 CFU/10 mL ≤10 CFU/100 mL 0 CFU/100 mL Excipients Polysorbate 80 0.026%-0.029% w/v 0.027%-0.034% w/v 0.024%-0.036% w/v Osmolality 281-290 mOsm/Kg 288-299 mOsm/Kg 267-327 mOsm/Kg

7.6.1. Color, Appearance, and Clarity

The appearance of DAC HYP drug substance is assessed by visually examining the color and clarity of the solution in direct light against a black background and white background without magnification. The solution is also evaluated for the presence of visible particles. The typical appearance of various lots of DAC HYP drug product is described in Table 17.

7.6.2. pH Determination

The pH of DAC HYP is determined in accordance with the U.S. Pharmacopeia Protocol No. (791). The pH ranges of various lots of DAC HYP drug product are summarized in Table 17.

7.6.3. Product Concentration by UV Spectroscopy

The concentration of DAC HYP is determined by UV spectroscopy. DAC HYP samples are diluted gravimetrically with buffer. The UV absorbance of each diluted sample solution is measured at 278 nm against a buffer blank. The protein concentration of the sample is calculated using the absorptivity coefficient for DAC HYP. The protein concentrations of various lots of DAC HYP drug product are summarized in Table 17.

7.6.4. N-Terminal Sequencing

DAC HYP 150 mg/mL lots were evaluated by N-terminal sequencing. The samples were analyzed using an automated Edman degradation sequencing instrument.

The expected amino acid sequence of the light chain through the first 15 residues, DIQMTQSPSTLSASV (SEQ ID NO:13), was confirmed for all samples.

The majority of heavy chain in DAC HYP is blocked by a pyroglutamate (pE) residue that will not produce an N-terminal heavy chain sequence. The next most prevalent N-terminal heavy chain sequence in DAC HYP begins with a valine, histidine, serine (VHS) sequence, resulting from the lack of processing the three terminal residues of the heavy chain signal peptide. Fourteen of the first fifteen N-terminal residues were confirmed for the VHS heavy chain sequence (VHSQVQLVQSGAEVK (SEQ ID NO: 14)) in all samples. The fourth residue, glutamine, could not be confirmed due to the large amount of glutamine detected from LC in the preceding sequencing cycle. Evidence of heavy chain with N-terminal glutamine was also present in all samples. This sequence is a result of the native N-terminal heavy chain glutamine residue not undergoing cyclization to the pyroglutamate form. The N-terminal sequencing results for the 150 mg/mL lots are consistent with the sequences predicted from the heavy and light chain coding sequences. Comparable results were obtained for the 100 mg/mL lots.

7.6.5. Heavy and Light Chain Mass Analysis

The molecular masses of the heavy chain and light chain of the DAC HYP 150 mg/mL lots and Reference Standard RS0801 were evaluated by liquid chromatography mass spectrometry (LC-MS) analysis. All lots were deglycosylated with the amidase PNGaseF, reduced with dithiothreitol, alkylated with iodoacetic acid, and separated by reversed phase chromatography. Theoretical heavy and light chain masses were calculated from the protein sequence. The observed masses of the samples were within 1 Da of the calculate masses, as shown in Table 18 below:

TABLE 18 Heavy and Light Chain Mass Results LC (Da) HC (pE, -K)(Da) HC (VHS, -K) (Da) Theoretical Mass 23,505 49,356 49,697 RS0801 23,505 49,357 49,696 Batch 150-1 23,505 49,357 49,696 Batch 150-2 23,504 49,357 49,696

As described in the preceding subsection, the two most prevalent forms of DAC HYP heavy chain are known to contain an N-terminal pyroglutamate (pE) residue or a valine, histidine, serine (VHS) sequence and lack C-terminal lysine. The molecular weights obtained for the two predominant heavy chain variants and the light chain in the 150 mg/mL lots were comparable to those of Reference Standard RS0801 and consistent with the masses predicted from the protein sequences.

Together with the peptide mapping results presented in the following subsection, the heavy and light chain mass results confirm the presence of the expected light chain and heavy chain primary structures in the DAC HYP 150 mg/mL lots.

7.6.6. Peptide Mapping

DAC HYP 150 mg/mL lots and Reference Standard RS0801 (DAC HYP produced from a 100 mg/mL drug substance lot manufactured at the 10,000 L scale) were evaluated using reversed phase HPLC peptide mapping. All lots were reduced with dithiothreitol, alkylated with iodoacetic acid, and enzymatically digested with trypsin. The resulting peptides were separated by reversed phase chromatography and detected by ultraviolet absorbance at 215 nm to generate peptide maps.

To verify the primary amino acid sequence, the peptide maps of the 150 mg/mL lots were compared to that of Reference Standard RS0801. Peptides corresponding to ninety eight percent of the expected heavy and light chain residues have previously been identified by mass spectrometry in the peptide map of Reference Standard RS0801. The residues that have not been accounted for in the peptide map are single amino acids or reside in very polar dipeptides, and are not expected to be retained by the reversed phase column. Masses consistent with pyroglutamate, glutamine, and the VHS sequence at the N-terminus of the heavy chain N-terminal peptide have been identified in the reference standard. DAC HYP contains a consensus site for N-linked glycosylation in the Fc portion of the heavy chain at Asn296 and masses consistent with linked complex core biantennary oligosaccharide structures have been identified for the peptide containing the Asn296 residue.

Peptide maps comparing the DAC HYP 150 mg/mL lots to Reference Standard RS0801 are shown in FIG. 10 (0 to 60 minutes), FIG. 11 (55 to 115 minutes), and FIG. 12 (110 to 170 minutes). The peptide maps of the DAC HYP 150 mg/mL lots are comparable to those of the reference standard and confirm the presence of the expected primary structure in the 150 mg/mL lots.

7.6.7. Circular Dichroism Spectroscopy

DAC HYP 150 mg/mL lots and Reference Standard RS0801 were analyzed by far ultraviolet circular dichroism spectroscopy (far-UV CD) to evaluate secondary structure. Prior to analysis samples were diluted with water to a final protein concentration of 0.2 mg/mL. Spectra were acquired from 195 to 260 nm using a 0.1 cm cell and the signal obtained was converted to molar ellipticity after buffer subtraction.

The overlaid far-UV CD spectra of DAC HYP 150 mg/mL lots Batch 1 and Batch 2 and Reference Standard RS0801 are shown in FIG. 13. The spectra of all lots show a positive band at approximately 202 nm and a negative band at approximately 217 nm. The negative band at 217 nm is characteristic of a P sheet conformation, the predominant conformation of IgG molecules. The far-UV CD spectra of the lots were similar, which is supportive of a uniform secondary structure among the DAC HYP 150 mg/mL lots and Reference Standard RS0801.

7.6.8. Ultraviolet Spectroscopy

DAC HYP 150 mg/mL lots and Reference Standard RS0801 were analyzed by ultraviolet (UV) spectroscopy to evaluate tertiary structure. Prior to analysis samples were diluted with formulation buffer (40 mM succinate, 100 mM sodium chloride, 0.03% polysorbate 80, pH 6.0) to a final protein concentration of 0.5 mg/mL. Spectra were acquired from 250 to 350 nm using a 1 cm path length quartz cuvette and normalized to an absorbance of 1.0 at 280 nm.

The overlaid zero-order and second derivative UV spectra (calculated from the smoothed zero-order data) are shown in FIGS. 14A and 14B, respectively. The zero-order and second derivative spectra of all lots evaluated were superimposable, which is supportive of a uniform tertiary structure among the DAC HYP 150 mg/mL lots Batch 1 and Batch 2 and Reference Standard RS0801.

7.6.9. Size Exclusion Chromatography

Size exclusion chromatography (SEC) was performed using a porous silica column with an aqueous mobile phase and ultraviolet absorbance detection at 280 nm. In particular, L test sample (20 mg/mL antibody in elution buffer) was analyzed at room temperature on a 7.8 mm×30 cm TSK G3000SW_(XL) column (Tosoh Biosciences, part no. 601342) equipped with a 0.5 m pre-column filter (Upchurch, part no. A-102X) using an isocratic gradient of elution buffer (200 mM KPO₄, 150 mM KCl, pH 6.9) at a flow rate of 1 mL/min.

As shown in FIG. 15A (full scale) and FIG. 15B (expanded scale), the chromatographic profiles of the DAC HYP 150 mg/mL lots were comparable to that of Reference Standard RS0801. All lots exhibited the same major peak corresponding to daclizumab monomer and a minor aggregate peak. Aggregate results for DAC HYP 150 mg/mL lots were comparable to those of DAC HYP 100 mg/mL lots as shown in Table 19 below:

TABLE 19 Percentage Aggregate Results 100 mg/mL Average 0.6 (n = 24 lots) Standard Deviation 0.1 Minimum 0.4 Maximum 0.8 150 mg/mL Batch 150-1 0.6 Batch 150-2 0.7

The 150 mg/mL lots and Reference Standard RS0801 were analyzed using SEC with multi-angle light scattering detection (SEC-MALS) to determine the molecular weight of the aggregate peak. For all lots, the molecular weight obtained for the aggregate peak was approximately 300 kDa, which is consistent with antibody dimer.

Aggregate formation in DAC HYP was monitored over an 18-month period. The level of aggregates in the formulation rose, but the percentage of aggregates plateaued and did not exceed approximately 1.5% when stored at 5° C. for 18 months (see FIG. 16). New data with new batches indicates that approximately 1.8-1.9% aggregates may appear at 6 weeks when stored at 5° C., but for all samples tested, less than 3% aggregates formed over a period of 5 years when stored at 5° C.

7.6.10. Sedimentation Velocity Analytical Ultracentrifugation

The monomer and aggregates in DAC HYP 150 mg/mL and 100 mg/mL lots were characterized using sedimentation velocity analytical ultracentrifugation (SV-AUC). The sedimentation coefficient value and relative abundance for the monomer and each of the aggregates are presented in Tables 20 and 21 below.

TABLE 20 Sedimentation Coefficient Values for Monomer and Aggregates (in Svedbergs) Monomer Dimer Trimer Tetramer 100 mg/mL Average 6.14 8.48 10.6 12.1 (n = 8 lots) Range 6.11-6.18 8.36-8.67 10.3-10.8 11.8-12.7 150 mg/mL Batch 6.16 8.52 10.4 11.6 150-1 Batch 6.13 8.33 10.3 11.9 150-2

TABLE 21 Relative Abundance of Monomer, Dimer, Trimer, and Tetramer % % % % Monomer Dimer Trimer Tetramer 100 mg/mL Average 97.5 1.8 0.4 0.2 (n = 8 lots) Range 96.7-98.5 1.3-2.2 0.2-0.7 0.1-0.4 150 mg/mL Batch 97.6 1.9 0.4 0.1 150-1 Batch 96.9 2.3 0.5 0.3 150-2

Monomer was the major component observed in each of the lots. The sedimentation coefficient of the monomer peak was highly consistent among the lots indicating that the conformation of the monomer is comparable between the 150 mg/mL and 100 mg/mL lots. The monomer content of the 150 mg/mL lots was comparable to that of the 100 mg/mL lots.

The predominant aggregate species in each of the lots had a sedimentation coefficient consistent with antibody dimer. This is consistent with SEC-MALS results, which indicate that the SEC aggregate peak is composed primarily of antibody dimer (see preceding subsection). Low levels of two larger aggregate species that had sedimentation coefficients consistent with trimer and tetramer were also observed in each of the lots by AUC. The dimer, trimer, and tetramer content of the 150 mg/mL lots was comparable to that of the 100 mg/mL lots.

7.6.11. Quantitative Reduced SDS-PAGE

Purity was determined by SDS-PAGE using 4-20% (typically 14%) tris-glycine gels with Colloidal blue stain. Samples were analyzed under reducing conditions with a sample load of 10 jag. Purity was calculated by dividing the sum of the heavy chain and light chain band area by the total band area as measured by densitometry.

As shown in Table 22 below, the 150 mg/mL lots are of high purity and comparable to the 100 mg/mL lots:

TABLE 22 Quantitative Reduced SDS-PAGE Results % Purity 100 mg/mL Average 96.4 (n = 7 lots) Standard Deviation 0.5 Minimum 95.8 Maximum 97.0 150 mg/mL Batch 150-1 97.3 Batch 150-2 97.3

7.6.12. Qualitative SDS-PAGE

Purity of DAC HYP was assessed by both reduced and non-reduced gel electrophoresis. Precast 14% or 8-16% Tris-glycine gels were used for the analysis. Aliquots from the two batches of 150 mg/mL DAC HYP formulation were compared to a reference batch, as previously described. Reduced and non-reduced gels analyzing the purity of DAC HYP are shown in FIG. 17. The band pattern of the 150 mg/mL lots was comparable to that of Reference Standard RS0801, with no new bands detected in the 150 mg/mL lots.

7.6.13. Cation Exchange Chromatography

The charge isoform distribution of the DAC HYP 150 mg/mL lots and 100 mg/mL lots were evaluated using cation exchange chromatography (CEX). CEX was performed using a nonporous, carboxylate functionalized, weak cation exchange column with detection at 220 nm. 100 μL of test sample (1 mg/mL antibody dissolved in Buffer A) was resolved at room temperature on a ProPac WCX-10 column (Dionex Coporation) equipped with a ProPac WCX-10G guard column (Dionex Corporation) using the following separation gradient (column is equilibrated with Buffer A):

Time (min.) % Buffer A % Buffer B Flow Rate (mL/min) 0.0 100 0 1 60.0 40 60 1 80.0 0 100 1 85.0 0 100 1 85.1 100 0 1 100.0 100 0 1 Buffer A = 15 mM sodium phosphate, pH 5.9 Buffer B = 250 mM NaCl, 15 mM sodium phosphate, pH 5

As shown in FIG. 18, the CEX chromatograms of the 150 mg/mL lots are consistent with those of Reference Standard RS0801, with no new charge isoforms detected in the 150 mg/mL lots. The five major isoforms present in the CEX chromatograms are due to heterogeneity at the heavy chain N-terminus and include: 1) two pyroglutamate residues (pE/pE); 2) one pyroglutamate residue and one glutamine residue (pE/Q); 3) one pyroglutamate residue and one VHS sequence (truncated VHS signal peptide preceding the N-terminal glutamine residue of the mature heavy chain) (pE/VHS); 4) one glutamine residue and one VHS sequence (Q/VHS); 5) two VHS sequences (VHS/VHS). C-terminal lysine (K) isoforms are also resolved and identified in FIG. 18. Each of the N-terminal isoforms described above may exist as different C-terminal isoforms (0, 1, or 2 K). Because of the complexity of the mixture, the C-terminal lysine isoforms are only clearly resolved and measurable for the major pE/pE and pE/VHS isoforms using the described method.

Quantitative N- and C-terminal isoform results are provided for the 150 mg/mL and 100 mg/mL lots in Tables 23 and 24, respectively, where the reported percentage is based upon the percentage of the area under the curve (AUC) of the specific peak as compared to the total AUC of all peaks:

TABLE 23 CEX Results-N-Terminal Isoforms % % % % % VHS/ pE/pE pE/Q pE/VHS Q/VHS VHS 100 mg/mL Average 38 11 40 7 5 (n = 24 Standard 2.8 0.8 2.0 1.1 3.8 lots) Deviation Minimum 31 9 34 4 1 Maximum 42 12 42 9 17 150 mg/mL Batch 150-1 31 9 31 12 17 Batch 150-2 32 9 31 11 17 pE/pE = Two Pyroglutamate Residues; pE/Q = One Pyroglutamate Residue, One Glutamine Residue; pE/VHS = One Pyroglutamate Residue, One Truncated Signal Peptide; Q/VHS = One Glutamine Residue, One Truncated Signal Peptide; and VHS/VHS = Two Truncated Signal Peptides.

TABLE 24 CEX Results - C-Terminal Isoforms % 0K % 1K % 2K 100 mg/mL Average 78 16 6 (n = 24 lots) Standard Deviation 2.5 1.7 1.0 Minimum 69 14 5 Maximum 80 22 10 150 mg/mL Batch A 73 19 8 Batch B 74 19 8 0K = No C-terminal lysine residue on either heavy chain 1K = C-terminal lysine residue on one heavy chain 2K = C-terminal lysine residue on both heavy chains

7.6.14. Oligosaccharide Mapping

The oligosaccharide distributions of the DAC HYP 150 mg/mL and 100 mg/mL lots were evaluated by oligosaccharide mapping. N-linked oligosaccharides were released enzymatically from heavy chain Asn296 using the amidase PNGaseF. The oligosaccharides were subsequently derivatized with a fluorescent label (in this case anthranilic acid) and separated from the antibody via a nylon membrane. The derivatized, cleaved N-linked glycans were resolved at 50° C. on a 250×4.6 mm polymeric-amine bonded Asahipak Amino NH₂P-504E column (5 m particle size, Phenomenex, cat. No. CHO-2628) with fluorescent detection, using the following elution gradient (using a sample injection volume of 100 μL; column is equilibrated with 85% Buffer A/15% Buffer B):

Time (min.) % Buffer A % Buffer B Flow Rate (mL/min) 0 85 15 1 2 85 15 1 10 80 20 1 60 55 45 1 70 5 95 1 75 5 95 1 76 85 15 1 90 85 15 1 Buffer A = 1% v/v tetrahydrofuran, 2% v/v acetic acid in acetonitrile Buffer B = 1% v/v tetrahydrofuran, 5% v/v acetic acid, 3% v/v triethylamine in water

Chromatograms comparing the 150 mg/mL lots to Reference Standard RS0801 are shown in FIG. 19. Oligosaccharide peaks constituting at least 1.0% of the total peak area are labeled and reported below in Table 25:

TABLE 25 Oligosaccharide Results % % % % G0-GlcNAc G0 Peak 3 G1 100 mg/mL Average 8.6 86.3 1.5 2.0 600 L (n = 22 lots) Minimum 6.9 84.6 1.4 1.5 Maximum 10.6 88.2 1.6 2.3 100 mg/mL Batch 100-A 11.2 82.3 1.7 3.2 10 kL (n = 2 lots) Batch 100-B 9.0 83.7 1.7 3.7 150 mg/mL Batch 150-1 7.3 85.6 1.6 3.8 10 kL (n = 2 lots) Batch 150-2 7.2 85.6 1.6 3.7 G0-GlcNAc: Biantennary core structure with fucose attached to the N-linked N-acetyl glucosamine, one N-acetyl glucosamine on one branch of the core structure and no terminal galactose. G0: Biantennary core structure with fucose attached to the N-linked N-acetyl glucosamine, one N-acetyl glucosamine on each branch of the core structure and no terminal galactose. G1: Biantennary core structure with fucose attached to the N-linked N-acetyl glucosamine, one N-acetyl glucosamine on each branch of the core structure and terminal galactose on one branch of the core structure.

All lots consist primarily of G0 and G0-GlcNAc (G0 lacking GlcNAc on one arm of the biantennary structure), which is representative of the DAC HYP process. Sialylated oligosaccharides elute at approximately 68 minutes and are below 1.0% in all lots tested. The uncharacterized oligosaccharide referred to as Peak 3 was present in similar abundance in all lots tested.

7.6.15. Oxidation

DAC HYP lots were evaluated for potential methionine oxidation, by monitoring oxidized and non-oxidized tryptic peptides present in the peptide maps. The peak areas of the non-oxidized and oxidized forms of each methionine containing peptide were determined using the mass spectra extracted ion chromatograms. For each methionine residue, the percent oxidized methionine was calculated by dividing the mass spectra peak area of the oxidized peptide by the sum of the peak areas of the oxidized and non-oxidized peptides.

As shown in Table 26 below, methionine oxidation results for the 150 mg/mL lots and five 100 mg/mL lots were comparable:

TABLE 26 Oxidation Results (LC = light chain; HC = heavy chain; M = methionine) Percent Oxidation Lots LC M4 LC M32 HC M34 HC M 81 HC M251 HC M427 100 mg/mL Batch 100-1 0.8 0.6 0.5 0.6 3.8 1.1 Batch 100-2 0.9 0.7 0.6 0.7 3.7 1.3 Batch 100-3 0.8 0.7 0.5 0.7 4.0 1.3 Batch 100-4 0.8 0.6 0.4 0.7 4.8 1.8 Batch 100-5 0.6 0.6 0.3 0.6 4.6 1.5 Average 0.8 0.7 0.5 0.7 4.2 1.4 Standard Deviation 0.1 0.1 0.1 0.1 0.5 0.3 Minimum 0.6 0.6 0.3 0.6 3.7 1.1 Maximum 0.9 0.7 0.6 0.7 4.8 1.8 150 mg/mL Batch 150-1 0.6 0.6 0.4 0.5 3.5 0.9 Batch 150-2 0.8 0.7 0.4 0.7 4.3 1.6

Heavy chain Met251 and Met427 are the most labile and exhibits the greatest degree of oxidation. Among the lots tested concurrently to evaluate comparability, oxidation levels for Met251 and Met427 did not exceed 4.8% and 1.8%, respectively.

7.6.16. Binding Potency (CD25 Binding)

DAC HYP 150 mg/mL and 100 mg/mL lots were evaluated for binding to the alpha subunit of the IL-2 receptor (CD25) via ELISA as a measure of potency as part of release testing. Microtiter plates were immobilized with soluble CD25 and incubated with varying amounts of DAC HYP. Bound DAC HYP was detected using a horseradish peroxidase-conjugated goat anti-human IgG antibody in tandem with 3,3′,5,5′-tetra-methyl benzidine substrate. Resulting absorbance values were plotted against the log₁₀ of DAC HYP concentration using a 4-parameter fit and percent relative potency values were generated using parallel line analysis.

Drug substance results are summarized the table below:

TABLE 27 Potency by ELISA Results Potency (% of Reference Standard) 100 mg/mL Average 101 (n = 24 lots) Standard Deviation 14 Minimum 78 Maximum 129 150 mg/mL Batch 150-1 86 Batch 150-2 105

The binding potency results of the 150 mg/mL lots were comparable to those of the 100 mg/mL lots.

7.6.17. Surface Plasmon Resonance (CD25 Binding)

Surface plasmon resonance analysis was performed to determine the affinity constant (K_(D)) for the binding interaction of DAC HYP to the alpha subunit of the IL-2 receptor (CD25).

Goat anti-human IgG Fc antibody was immobilized on a chip surface to capture DAC HYP samples, after which soluble CD25 was injected at various concentrations in duplicate over captured DAC HYP using an automated method. Binding data were collected and corrected using a reference flow cell and buffer blank, and fit with BIA Evaluation software using a 1:1 Langmuir model to obtain equilibrium constants.

Results for DAC HYP 150 mg/mL lots and Reference Standard RS0801 are summarized in Table 28:

TABLE 28 Surface Plasmon Resonance Results Lots Ka (1/M*s) Kd (1/s) KD (M) % RS KD RS0801 (“RS”) 3.3 × 10⁵ 1.5 × 10⁻⁴ 4.4 × 10⁻¹⁰ Not Applicable Batch 150-1 3.3 × 10⁵ 1.5 × 10⁻⁴ 4.6 × 10⁻¹⁰ 96 Batch 150-2 3.4 × 10⁵ 1.5 × 10⁻⁴ 4.4 × 10⁻¹⁰ 100

The association constant (k_(a)), dissociation constant (k_(d)), and affinity constant (K_(D)) values of the 150 mg/mL lots were comparable to those of Reference Standard RS0801.

7.6.18. Functional Potency

DAC HYP 150 mg/mL and 100 mg/mL lots were evaluated for functional potency as part of release testing. The functional potency assay measures the inhibition of IL-2 induced T-cell proliferation by binding of DAC HYP to the alpha subunit of the IL-2 receptor (CD25). In the presence of IL-2, varying amounts of DAC HYP were incubated with KIT-225 K6 cells (Hori et al., 1987, Blood 70:1069-1072) expressing the IL-2 receptor. Inhibition of T-cell proliferation by DAC HYP was subsequently detected using alamar blue. Resulting fluorescence values were plotted against the log₁₀ of DAC HYP concentrations using a 4-parameter fit and percent relative potency values were generated using parallel line analysis.

The functional potency results are summarized in Table 29:

TABLE 29 Functional Potency Results Potency (% of Reference Standard) 100 mg/mL Average 98 (n = 24 lots) Standard Deviation 12 Minimum 73 Maximum 121 150 mg/mL Batch 150-1 101 Batch 150-2 95

The functional potency results of the 150 mg/mL lots were comparable to those of the 100 mg/mL lots.

7.6.19. Antibody Dependent Cellular Cytotoxicity

Two lots of DAC HYP 150 mg/mL formulations were evaluated relative to that of Reference Standard RS0801 100 mg/mL DAC HYP.

IL-2 receptor expressing KIT-225 K6 cells were labeled with ⁵¹Cr, and subsequently incubated with DAC HYP. Human effector cells (PBMC) were added in varying amounts to achieve different effector to target cell (KIT-225 K6) ratios. Fc receptor bearing monocytes interact with the DAC HYP Fc domain and subsequently cause target cell lysis. The degree of cytotoxicity was determined by measuring the release of ⁵¹Cr from target cells and was expressed as a percentage of maximum cell lysis.

PBMCs from multiple donors were utilized for each sample. For each donor, the percent ADCC activity of the sample was calculated relative to that of Reference Standard RS0801 based on percent cytotoxicity. ADCC results are summarized in Table 30 below:

TABLE 30 ADCC Results % Cytotoxicity (Relative to Reference Standard RS0801) Standard Lot Donor #1 Donor #2 Donor #3 Average Deviation Batch 150-1 93 79 76 83 9 Batch 150-2 93 92 80 88 7

Response curves for the 150 mg/mL lots, Reference Standard RS0801, positive and negative control antibodies and a control without antibody (for Antibody Independent Cellular Cytotoxicity or AICC) are shown in FIG. 20.

The ADCC activity of the 150 mg/mL lots was comparable to that of Reference Standard RS0801.

7.6.20. Residual Protein A

Residual Protein A may be determined by an ELISA method, where standards, sample controls, a plate blank, and test samples are diluted with a denaturing buffer and placed into a boiling water bath to dissociate Protein A and denature and precipitate daclizumab. After boiling, standards, controls, and samples are cooled, centrifuged, and added to a micro-titer plate coated with polyclonal anti-Protein A capture antibody. Residual Protein A present in the samples is then detected using a biotinylated anti-Protein A antibody in tandem with streptavidin alkaline phophatase and P-nitrophenyl phosphate (PNPP) substrate. The plate is analyzed in a spectrophotometric plate reader and a log-log standard curve is generated, against which the concentration of Protein A is determined. Test sample results are reported in parts per million (ppm) units. Parts per million results are calculated by dividing the ng/mL Protein A result by the antibody concentration of the test sample in mg/mL.

7.6.21. DNA Content

Detection of mouse DNA is determined at a contract laboratory using a quantitative polymerase chain reaction (Q-PCR) test method. In the method, the sample is subjected to DNA extraction. The sample extract is then analyzed by Q-PCR using mouse specific primers and probe to amplify a specific fragment of a repetitive element of mouse DNA. Amplification of the DNA results in a fluorescence signal that is detected. The DNA in the sample is quantitatively measured by comparison to a standard curve generated using known amounts of mouse DNA. Results are expressed in picograms of DNA per milligram of antibody. The average DNA content in various lots of DAC HYP drug product are summarized in Table 17.

7.6.22. Host Cell Proteins (HCP)

Residual host cell proteins in the product are quantified using a commercially available kit. An affinity purified goat polyclonal antibody to NS0 cell lysate is used for both the capture and detection of NS0 HCP. The HCP standard is produced by collecting cell free harvest material from a mock production run. A standard curve is prepared using an HCP working standard and samples containing HCP are serially diluted to target the range of the standard curve. Standards, sample controls, and test samples are added to an anti-NS0 HCP polyclonal antibody coated plate. Host cell proteins are then detected with an anti-NS0 HCP polyclonal antibody conjugated to horseradish peroxidase (HRP) in tandem with 3,3′,5,5′-tetra-methyl benzidine (TMB) substrate. The plate is then analyzed in a spectrophotometric plate reader and a four parameter curve fit is generated to quantitate the amount of HCP in the samples.

The results for the NS0 HCP ELISA assay are reported in parts per million (ppm) units. Parts per million results are calculated by dividing the ng/mL HCP result by the antibody concentration in mg/mL. The average HCP of various lots of DAC HYP drug product are summarized in Table 17.

7.6.23. Polysorbate 80 Concentration

Polysorbate 80 is quantitated using a spectrophotometric method that is based on the formation of a colored cobaltthiocyanate complex with polysorbate 80. A standard curve is constructed using a series of polysorbate 80 standards. The polysorbate 80 concentration in the sample is determined from the standard curve. The ranges of polysorbate concentrations of various lots of DAC HYP drug product are summarized in Table 17.

7.6.24. Osmolality

Osmolality is measured using a vapor pressure depression osmometer. Prior to sample analysis the osmometer is calibrated using osmolality standards that bracket the expected osmolality of the sample. The osmolality ranges of various lots of DAC HYP drug product are summarized in Table 17.

7.6.25. Conclusions

The physicochemical and biological analyses conducted provide a comprehensive evaluation of DAC HYP 150 mg/mL and DAC HYP 100 mg/mL formulations. The physicochemical and biological characteristics of all lots tested to date are comparable.

For all DAC HYP lots, the first 15 amino acids of the heavy and light chains determined by N-terminal sequencing, peptide maps and molecular weight analyses were consistent with the daclizumab gene sequences.

The aggregate levels and size distribution of aggregate species in all 150 mg/mL and 100 mg/mL lots tested, as determined by SEC-MALS and SV-AUC were comparable, as were their purity as tested by gel electrophoresis.

The charge isoform distribution of the 150 mg/mL lots was similar to that of the 100 mg/mL lots, with only slight differences in the relative amounts of the pE/VHS (150 mg/mL lots=31% pE/VHS; 100 mg/mL lots=34 to 42% pE/VHS) and Q/VHS (150 mg/mL lots=11 to 12% Q/VHS; 100 mg/mL lots=4 to 9% Q/VHS) N-terminal isoforms. The characteristics of DAC HYP are as follows:

N-Terminal Isoforms by CEX:

pE/pE: 31-46% pE/Q: 7-12% pE/VHS: 31-42%

Q/VHS: 3-12% VHS/VHS: 1-17% C-Terminal Isoforms by CEX: 0K: 53-80% 1K: 14-28% 2K: 5-19%

The N-linked glycan distribution of DAC HYP is as follows:

G0-GlcNAc: 7.2-14.6% G0: 80.9-88.2% Peak 3: 1.3-1.7% G1: 1.4-3.8

The oxidation levels measured for DAC HYP were low.

DAC HYP is biologically active, as confirmed in ELISA and surface plasmon resonance CD25 binding experiments, as well as functional to inhibit IL-2 induced T-cell proliferation. DAC HYP is also characterized by a low level of aggregation upon storage.

7.7. DAC HYP Stability

High concentration DAC HYP formulations are stable upon storage. The following tables provide stability data for 150 mg/mL DAC HYP drug substance lots.

Table 31 below provides stability data following storage in 50 mL bags at the recommended conditions (2-8° C.). Table 32 below provides accelerated stability data storage in 50 mL bags at 23-27° C. Table 33 below provides stressed stability data.

TABLE 31 Primary Real-Time Stability of DAC HYP Drug Substance, 150 mg/mL (Batch A) Time Point Color and SEC % SEC % Binding Functional (Months) Appearance pH Conc. CEX Main Peak Aggregate % Purity* Potency Potency 0 Pass^(a) 6.0 148 Pass^(b) 99.4 0.6 97.3 86 101 1 Pass 6.0 149 Pass 99.0 1.0 97.0 90 94 2 Pass 6.0 149 Pass 99.0 1.0 96.9 91 108 3 Pass 6.0 149 Pass 98.9 1.1 97.3 102 104 6 Pass 6.0 149 Pass 98.8 1.2 97.5 94 79 9 Pass 6.0 151 Pass 98.5 1.4 97.3 107 110 12 Pass 6.0 152 Pass 98.4 1.5 95.7 96 98 ^(a)Pass criteria: Colorless, clear to slightly opalescent liquid, essentially free of visible particles. ^(b)Pass criteria: Chromatogram profile consistent with reference. *SDS-PAGE (colloidal blue stain)

TABLE 32 Primary Accelerated Stability of DAC HYP Drug Substance, 150 mg/mL (Batch A) Time Point Color and SEC % SEC % Binding Functional (Months) Appearance pH Cone. CEX Main Peak Aggregate % Purity* Potency Potency 0 Pass^(a) 6.0 148 Pass^(b) 99.4 0.6 97.3 86 101 1 Pass 6.0 150 Pass 98.5 1.4 97.0 93 111 2 Pass 6.0 152 Pass 98.3 1.5 96.0 110 105 3 Pass 6.0 153 Pass 98.2 1.6 96.0 100 108 6 Pass 6.0 155 Pass 97.7 1.8 95.2 94 109 ^(a)Pass criteria: Colorless, clear to slightly opalescent liquid essentially free of visible particles. ^(b)Pass criteria: Chromatogram profile consistent with reference. *SDS-PAGE (colloidal blue stain)

TABLE 33 Primary Stressed Stability of DAC HYP Drug Substance, 150 mg/mL (Batch A) Time Point Color and SEC % SEC % Binding Functional (Months) Appearance pH Cone. CEX Main Peak Aggregate % Purity* Potency Potency 0 Pass 6.0 148 Pass 99.4 06 97.3 86 101 1 Pass 6.0 153 Pass 97.8 1 8 94.8 85 93 2 Pass 6.0 159 Pass 97. 1 22 90.0 102 108 ^(a)Pass criteria: Colorless, clear to slightly opalescent liquid, essentially free of visible particles. ^(b)Pass criteria: Chromatogram profile consistent with reference. *SDS-PAGE (colloidal blue stain)

7.8. Comparison Between Different Forms of Daclizumab

Hoffman-La Roche, Inc. (“Roche”) manufactured an intravenous formulation of a daclizumab marketed as ZENAPAX™ for treatment of allograft rejection that has been discontinued. DAC Penzberg is a 100 mg/ml subcutaneous formulation of daclizumab used in clinical trials by PDL BioPharma (see CHOICE study described in Section 7.9.1 below).

A comparison between the DAC HYP, ZENAPAX DAC and DAC Penzberg formulations is shown in Table 34. In the table, the formulation buffer is the buffer the DAC was diafiltered into to yield the ultimate formulation. Accordingly, the noted concentrations are nominal concentrations:

TABLE 34 DAC HYP vs. ZENAPAX DAC and DAC Penzberg Material Formulation Conc. (mg/mL) ZENAPAX DAC 67 mM sodium phosphate 5 79 mM sodium chloride 0.02% polysorbate 80 pH 6.9 DAC Penzberg 40 mM succinate 100 DAC HYP 100 mM sodium chloride 100 or 150 0.03% polysorbate 80 pH 6.0

Various characteristics DAC HYP were compared to those of ZENAPAX DAC and DAC Penzberg.

A comparison between the glycosylation of DAC HYP vs. ZENAPAX DAC is shown in FIG. 21. The analysis of exemplary lots of the three forms of daclizumab is set forth in Table 35:

TABLE 35 Comparison of glycosylation of DAC HYP vs. ZENAPAX DAC vs. DAC Penzberg Source G0 (%) G1 (%) G2 (%) G0-GlcNAc ZENAPAX DAC 44 27 6 6 DAC Penzberg 39 31 8 3 DAC HYP 84 2 <1 9

DAC HYP also has significantly lower levels of mannose glycosyls (e.g., Man5, Man6, Man7) and lower levels of sialylated glycosyls than ZENAPAX DAC (see, e.g., FIG. 21).

Antibody dependent cell-mediated cytotoxicity (ADCC) is an in vitro assay that can be used to assess the Fc dependent activity and the potential cytotoxic effects of antibody-target binding. Using peripheral blood mononuclear cells (PBMC) from six healthy donors as effector cells and the CD25-expressing KIT225/K6 cell line as the target cells, the ADCC activity of the various daclizumab preparations was assayed in both a variable an effector to target cell ratio format or a variable antibody concentration format.

For the variable effector to target cell ratio format, ⁵¹Cr-labeled KIT225/K6 cells (12,500 cells/well) were pre-incubated with 1 μg/mL of antibody (final concentration) for 30 minutes at 4° C. in V-bottom 96-well plates in a volume of 100 μL of ADCC Assay Medium (containing 435 mL RPMI-1640; 5.0 mL L-glutamine; 50 mL heat inactivated fetal bovine serum; 500 μl 1000×2-mercaptoethanol; 5.0 mL of penicillin-streptomycin (100×); and 5.0 mL of HEPES (1 M stock) per 500 mL); control cells were incubated in ADCC Assay Medium in the absence of antibody for subsequent determination of antibody-independent ⁵¹Cr release.

The PBMC (effectors) were diluted serially in ADCC Assay Medium in a separate 96-well polypropylene plate, yielding cell concentrations per 100 μL of 6.25×10⁵ cells, 3.13×10⁵ cells, 1.56×10⁵ cells, 7.81×10⁴ cells, or 3.91×10⁴ cells. A volume of 100 μL per well of PBMC suspension was added to the plates containing ⁵¹Cr-labeled KIT225/K6 and antibodies, yielding Effector to Target (E:T) ratios of 50:1, 25:1, 12.5:1, 6.25:1 and 3.13:1. In addition, a volume of 100 μL per well of ADCC Assay Medium alone (no effector) was added to ⁵¹Cr-labeled KIT225/K6+mAbs, to determine spontaneous release of ⁵¹Cr. The assay plates were spun at 50 RCF for 2 minutes and incubated at 37° C. in a 7.5% CO₂ incubator for 4 hours.

Thirty minutes before the end of the 4-hour incubation, a volume of 25 μL of 8% TritonX-100 was added to the appropriate control wells to determine maximum release of ⁵¹Cr from target cells. Upon completion of the 4-hour incubation, the plates were spun at 350 RCF for 5 minutes and a volume of 100 μL of supernatant from each well was transferred to mini-tubes. Each mini-tube was inserted into a scintillation vial and counted for 1 minute in a Beckman Gamma 5500B counter, or equivalent.

For the variable antibody concentration format, ⁵¹Cr-labeled KIT225/K6 cells (12,500 cells/well; targets) were pre-incubated with various doses of antibodies (5, 1, 0.2, 0.04, 0.008, and 0.0016 μg/mL) of mAbs (final concentration) for 30 minutes at 4° C. in V-bottom 96-well plates in a volume of 100 μL of ADCC Assay Medium. The control cells were incubated with ADCC Assay Medium alone (no mAb) for subsequent determination of antibody-independent ⁵¹Cr release.

The PBMC (effectors) were diluted serially in ADCC Assay Medium, in a separate 96-well polypropylene plate to a concentration of 3.13×10⁵ cells/100 μL. A volume of 100 μL per well of PBMC suspension was added to the plates containing ⁵¹Cr-labeled KIT225/K6+mAbs, yielding an Effector to Target (E:T) ratio of 25:1. In addition, a volume of 100 μL per well of ADCC Assay Medium alone (no effector) was added to ⁵¹Cr-labeled KIT225/K6+mAbs, to determine spontaneous release of ⁵¹Cr. The assay plates were spun at 50 RCF for 2 minutes and incubated at 37° C. in a 7.5% CO₂ incubator for 4 hours.

Thirty minutes before the end of the 4-hour incubation, a volume of 25 μL of 8% TritonX-100 was added to the appropriate control wells to determine maximum release of ⁵¹Cr from target cells. Upon completion of the 4-hour incubation, the plates were spun at 350 RCF for 5 minutes and a volume of 100 μL of supernatant from each well was transferred to mini-tubes. Each mini-tube was inserted into a scintillation vial and counted for 1 minute in a Beckman Gamma 5500B counter, or equivalent.

The ADCC results are shown in FIG. 22A (variable effector to target cell ratio format) and FIG. 22B (variable antibody concentration format). These data show that the maximal ADCC activity achieved with DAC HYP tested at graded concentrations was approximately 30-40% lower than the activity elicited by the same concentration of ZENAPAX DAC and DAC Penzberg.

A comparison of the charge isoform profiles (corresponding to the N terminal variants) of DAC HYP vs. ZENAPAX DAC vs. DAC Penzberg is shown in FIG. 23.

7.9. DAC HYP Clinical Trials

7.9.1. CHOICE Study

The CHOICE trial was a phase 2, randomized, double-blind, placebo-controlled trial of daclizumab added to interferon beta therapy in 230 patients with relapsing MS. The trial tested two dosing regimens of 100 mg/ml DAC Penzberg (see Section 7.8 above for a description of the product) administered as a subcutaneous injection: 1 mg/kg daclizumab administered every four weeks and 2 mg/kg daclizumab administered every two weeks. Results of the study showed that the addition of daclizumab, administered at 2 mg/kg every two weeks to interferon beta therapy, significantly reduced new or enlarged gadolinium-enhancing lesions at week 24, when compared to interferon beta therapy alone.

The results of the CHOICE study are described in Wynn et al., 2010, Lancet Neurol. 9(4):381-90. Daclizumab treatment was generally well-tolerated. Common adverse events were similar in all treatment arms. Grade 3 adverse events were observed in 24 percent of DAC/IFNß-treated patients and 14 percent of placebo/IFNß-treated patients. The most frequent grade 3 adverse events were infections, which occurred in 7 percent of DAC/IFNß-treated patients and 3 percent of placebo/IFNβ-treated patients. There were no opportunistic infections or deaths, and all infections resolved with standard therapies.

The CHOICE trial demonstrated that, in MS patients on a background of IFNβ-a1 therapy, daclizumab was well-tolerated and caused a dose dependent reduction in new/enlarged gadolinium-enhancing (Gd+) lesions by 72% compared to IFNβ-a1 alone. Clinical efficacy was associated with a marked expansion of immunoregulatory CD56^(bright) natural killer (NK) cells.

7.9.2. SELECT Study

A randomized, double-blind, placebo-controlled dose ranging study (SELECT) was conducted to determine the safety and efficacy of two different dosage levels of DAC HYP.

Overview.

The study was conducted at 76 centers in the Czech Republic, Germany, Hungary, India, Poland, Russia, Ukraine, and the United Kingdom. The care of each patient involved a treating neurologist, a treating nurse (or study coordinator), an examining neurologist, an MRI technician, and a pharmacist (or authorized designee). A centralized Interactive Voice Response System was used for randomization across all sites. A protocol-defined interim futility analysis was performed after 150 patients completed the Week 24 visit.

Patients.

Eligibility criteria included patients 18-55 years of age with clinically definite relapsing remitting multiple sclerosis (according to 2005 McDonald criteria #1-4; see, Polman et al, 2005 Ann Neurol 58:840-846), a baseline Expanded Disability Status Scale (EDSS) of 0-0.50 (Kurtzke, 1983, Neurology 33(11):1444-52) and at least one MS relapse in the 12 months before randomization or one new Gd+ lesion on brain MRI performed within the 6 weeks prior to randomization, were randomized to receive either DAC HYP (150 mg or 300 mg) or placebo as a subcutaneous injection once every 4 weeks for 52 weeks. Patients with child-bearing potential needed to practice effective contraception.

Patients were excluded if they had primary-progressive, secondary-progressive, or progressive-relapsing MS, a history of malignancy, severe allergic or anaphylactic reactions or known drug hypersensitivity, or other significant medical conditions that, in the opinion of the investigator, would preclude administration of DAC HYP. Additional exclusion criteria included previous treatment with DAC HYP or ZENAPAX™, total lymphoid irradiation, cladribine, mitoxantrone, T-cell or T-cell receptor vaccination or any therapeutic mAb, except natalizumab or rituximab. At the time of randomization, patients could not have received treatment with cyclophosphamide or rituximab within the previous year; natalizumab, cyclosporine, azathioprine, methotrexate, intravenous immunoglobulin, plasmapheresis or cytapheresis within the previous 6 months; or live virus vaccine, treatment with glatiramer acetate, IFNβ, interferon-alpha, 3 months before randomization; or corticosteroids, 4-aminopyridine or related products within the previous 30 days.

Characteristics of the groups were as follows:

DAC HYP Placebo 150 mg 300 mg (n = 204) (n = 208) (n = 209) Demographics Age, years, mean (SD) 36.6 (9.0) 35.3 (8.9) 35.2 (8.7) Gender, female, n (%) 128 (63) 140 (67) 134 (64) Race, White, n (%) 197 (97) 202 (97) 200 (96) MS disease characteristics No prior MS therapy, n (%)* 155 (76) 155 (75) 162 (78) Years since MS diagnosis, 4.1 (2.0) 4.5 (3.0) 3.7 (3.0) mean (median) Number relapses past year, 1.3 (0.6) 1.4 (0.7) 1.3 (0.7) mean (SD) Baseline EDSS, mean (SD) 2.7 (1.2) 2.8 (1.2) 2.7 (1.2) MRI brain lesions ≥1 Gd+ lesions, n (%)** 90 (44) 106 (51) 74 (35) No. Gd+ lesions, mean (SD) 2 (4.5) 2.1 (3.5) 1.4 (3.3) No. T2 hyperintense lesions, 40 (32) 45 (35) 36 (31) mean (SD) DAC, daclizumab; HYP, high yield process; SD, standard deviation; MS, multiple sclerosis; EDSS, Expanded Disability Status Scale; Gd+, gadolinium-enhancing. *Patients who had not received prior MS treatment with the exception of steroids. **Placebo n = 203, DAC HYP 150 mg n = 206, DAC HYP 300 mg n = 206 (all p values were >0.05 for intergroup comparison).

Endpoints.

The primary objective of this study was to determine whether DAC HYP monotherapy reduced MS relapses as defined by the annualized relapse rate (ARR) at Week 52. Relapses were defined as new or recurrent neurologic symptoms (not associated with fever or infection), lasting >24 hours, and accompanied by new neurological findings upon assessment by the examining neurologist. An Independent Neurology Evaluation Committee (INEC), consisting of three blinded MS neurologists, evaluated all suspected relapses to adjudicate whether the protocol definition of MS relapse was satisfied. Only INEC approved relapses were included in the primary analysis.

The secondary objectives were to determine whether DAC HYP was effective in reducing the number of cumulative new Gd+ lesions on brain MRI scans performed at Weeks 8, 12, 16, 20 and 24 in a subset of patients; reducing the number of new or newly-enlarging T2 hyperintense lesions at Week 52; reducing the proportion of relapsing patients between baseline and Week 52; and improving quality of life (QoL), as measured by the change from baseline in the 29-item Multiple Sclerosis Impact Scale (MSIS-29) (Hobart et al., 2001, Brain 124(Pt 5):962-73) physical impact score at Week 52. Confirmed disability progression was assessed by change in EDSS score between baseline and Week 52 (1.0-point increase in EDSS for baseline EDSS ≥1.0 or 1.5 point increase for baseline EDSS=0 that was sustained for 12 weeks). EDSS evaluations were conducted every 12 weeks, and at Weeks 20, 52, 60 and 72.

Additional QoL endpoints were the subject's global assessment of well being, as assessed by the EQ-Visual Analogue Scale (EQ-VAS) (EuroQol-a new facility for the measurement of health-related quality of life, 2011, Accessed 17.11.11, at http://www.euroqol.org/); and change in the EQ-5D health survey (EuroQol-a new facility for the measurement of health-related quality of life, 2011, Accessed 17.11.11, at http://www.euroqol.org/); 12-item short form health survey SF-12 (Ware et al., 1996, Medical Care 34(3):220-33) and the MSIS-29 psychological scale at Week 52 (Hobart et al., 2001, Brain 124(Pt 5):962-73).

Additional MRI endpoints were the number of Gd+ lesions at Week 52, the volume of total and new or newly enlarging T2 hyperintense lesions at Weeks 24 and 52, the volume of total and new T1 hypointense lesions “black holes” (defined as lesions that were iso/hypointense to gray matter and that did not enhance after gadolinium administration) at Weeks 24 and 52, and the percentage change in whole brain volume assessed by the SIENA method (Smith et al., 2001, J Comput Assist Tomogr 25(3):466-75).

Lymphocyte subsets were measured at multiple time points using a validated FACS assay. CD56^(bright) NK cells were defined as CD3⁻/CD16⁺/CD56^(bight) lymphocytes. Immunogenicity to DAC HYP was assessed using a standard ELISA to screen for anti-drug antibodies and a cellular assay was then used to test for neutralizing antibodies on all positive samples.

Statistical Analyses.

A sample size of approximately 600 patients was selected to have 90% power to detect a 50% reduction in the ARR between a DAC HYP treatment group and the placebo group, estimated from simulations assuming a negative binomial distribution with a 10% drop out rate, a 5% type 1 error rate and a two sided test. The ARR in the placebo group was assumed to be 0.476, based on recently completed trials in RRMS subjects. All reported p-values are two-tailed.

The primary analysis evaluated differences in the ARR between each DAC HYP group versus placebo. Relapses that occurred after rescue treatment with alternative MS medication were censored. The difference was evaluated using a negative binomial regression model adjusting for the number of relapses in the year before study entry, baseline EDSS (EDSS <2.5 versus EDSS >2.5) and baseline age (≤35 versus >35 years). Secondary analyses tested for treatment differences using negative binomial regression (number of new Gd+ lesions between weeks 8 and 24; number of new or newly enlarging T2 hyperintense lesions), a Cox proportional hazards model (time to first relapse, time to disease progression), and an analysis of variance model (change in EDSS, volume of new or newly enlarging T2 lesions, volume of new T1 hypointense lesions, QoL) and a proportional odds model (number of new Gd+ lesions at Week 52). The proportion of patients who were relapse-free was estimated from the Kaplan-Meier survival curve distribution.

For the cumulative number of new Gd+ lesions between Weeks 8 and 24, if a patient missed 1 or 2 consecutive scans, or all scans, the last non-baseline observation was carried forward, or the mean number of lesions within each treatment group was used, respectively. For other MRI endpoints, missing data was imputed using the mean within the treatment group. For MSIS-29, if the patient was missing <10 items, the mean of the non missing items was used to impute the score. For patients missing ≥10 items and for other QoL measures, a random slope and intercept model was used to estimate missing data.

Statistical testing for efficacy endpoints utilized separate comparisons of the DAC HYP 300 mg group versus placebo and the DAC HYP 150 mg group versus placebo. A sequential closed testing procedure was used to control the overall Type I error rate due to multiple comparisons.

Efficacy analyses were evaluated in the intent-to-treat (ITT) population which included all patients who underwent randomization. However, 21 patients from a single study center were prospectively excluded from the ITT population prior to study completion due to evidence of incorrect dosing at the center, which was identified prior to study completion(all patients at the center were receiving active treatment). In a sensitivity analysis, all primary and secondary efficacy analyses were repeated using all randomized patients. All safety analyses were based on the safety population, which was defined as all patients who received at least one dose of study medication and who had at least one post randomization assessment.

A preplanned futility analysis was performed after 150 subjects completed the Week 24 visit, to provide an opportunity to stop if the hypothesized effects of DAC HYP were not evident. Since efficacy may change over the duration of the study there was no plan to stop the study early for evidence of superiority at the time of the futility analysis. Futility was assessed by estimating separately the conditional power for both the cumulative number of new Gd+ lesions between weeks 8 and 24 and the ARR endpoint for each dose group. The Safety Monitoring Committee reviewed the data at the time of the analysis and based on the overall consistency of the data and the assessment of risk benefit recommended to continue the study.

7.9.3. Summary Results.

Eligible participants were randomized from Feb. 15, 2008 to May 14, 2010. Baseline characteristics were similar across the three treatment groups, although there was a trend for patients in the DAC HYP 150 mg group to have more T2 and Gd+T1 lesions than those in the DAC HYP 300 mg group. Across all randomized patients, a total of 577 (93%) completed the treatment period with similar proportions of DAC HYP and placebo-treated patients completing the study.

Detailed Results. Clinical Efficacy.

The ARR at 52 weeks (primary endpoint) was lower for patients randomized to DAC HYP 150 mg (0.21) or 300 mg (0.23), compared with placebo (0.46; Table 36), representing a 54% reduction versus placebo with DAC HYP 150 mg (95% CI, 31% to 69%, p<0.0001), and a 50% reduction versus placebo for DAC HYP 300 mg (95% CI, 26% to 66%, p=0.0002; Table 36). Over 52 weeks, the proportion of relapsing patients was reduced in the DAC HYP 150 mg (19%) and 300 mg (20%) groups relapsed versus 36% in the placebo group (p≤0.001 for both comparisons) (Table 36). Compared with placebo, the risk of 3-month sustained disability progression at Week 52 was reduced by 57% (Hazard ratio=0.43; 95% CI, 0.21 to 0.88; p=0.021) in the DAC HYP 150 mg and by 43% (Hazard ratio=0.57; 95% CI, 0.30 to 1.09; p=0.091) in the DAC HYP 300 mg group.

A relative 4.0 improvement in the MSIS-29 physical score at Week 52 was observed for DAC HYP 150 mg versus placebo with a less marked change in the DAC HYP 300 mg patients, (p<0.0008 and p=0.1284 vs. placebo, respectively; Table 36). Similar improvements on other measures of health-related quality of life including measures of both physical and psychological function and overall health were also observed (Table 36).

TABLE 36 Clinical and MRI End Points by Treatment Group P Value DAC HYP DAC HYP 150 DAC HYP 300 Placebo (n = 196) 150 mg (n = 201) 300 mg (n = 203) mg vs. Placebo mg vs. Placebo Clinical Number of Relapses 0 127 (65) 163 (81) 163 (80) 1 52 (27) 33 (16) 33 (16) 2 15 (8) 5 (2) 5 (2) 3 2 (1) 0 1 (<1) ≥3 0 0 0 <0.0001 0.0002 ARR over 52 weeks (95% CI) 0.46 (0.37-0.57) 0.21 (0.16-0.29) 0.23 (0.17-0.31) Rate ratio (95% CI)* 0.46 (0.32-0.67) 0.50 (0.35-0.72) <0.0001 0.0002 % patients who relapsed at 52 weeks 36 19 20 Hazard ratio (95% CI)† 0.45 (0.30-0.67) 0.50 (0.35-0.72) <0.0001 0.0003 Disability progression at 3-months, % 13.3 5.9 7.8 Rate ratio (95% CI)†† 0.43 (0.21-0.88) 0.57 (0.30-1.09) 0.021 0.0905 Mean change EDSS from baseline to wk 52 0.09 −0.08 0.05 0.0102 0.4874 MRI New Gd+ lesions between Weeks 8-24 # patients with data¶ 104 101 102 Mean no. (95% CI)∥ 4.8 (3.6-6.4) 1.5 (1.1-2.0) 1.0 (0.7-1.5) <0.0001 <0.0001 % reduction versus placebo (95% CI) 69 (52.4-80.4) 78 (66-86.4) New Gd+ lesions at Week 52 No. of patients with data 195 199 200 Mean no. 1.4 0.3 0.2 <0.0001 <0.0001 Odds Ratio (95% CI) 0.15 (0.09-0.25) 0.12 (0.07-0.20) <0.0001 <0.0001 New/newly enlarging T2 hyperintense lesions at Week 52 # patients 195 199 200 Mean no. (95% CI)** 8.1 (6.7-9.9) 2.4 (2.0-3.0) 1.7 (1.4-2.2) <0.0001 <0.0001 % reduction versus placebo, (95% CI) 70 (59.4-77.9) 79 (71.3-84.2) Percentage change from baseline in volume T2 hyperintense lesions at Week 52 # patients 193 198 197 Mean (SD)** 27.3 (107.8) −11.1 (12.1) −12.5 (12.5) <0.0001 <0.0001 Percentage change from baseline in volume of new T1 hyperintense lesions at Week 52 # patients 195 199 200 Mean (SD)** 218.7 (400.2) 116.7 (276.6) 54.8 (153.1) <0.0001 <0.0001 Percentage mean change in whole brain volume to Week 24 # patients 194 198 200 Mean (SD)∞ −0.32 (0.729) −0.41 (0.769) −0.31 (0.678) 0.0635 0.6261 Percentage mean change in whole brain volume Week 24 to Week 52 # patients 194 198 200 Mean (SD)∞ −0.74 (0.90) −0.79 (0.83) −0.70 (0.91) 0.3263 0.4117 Quality of life MSIS-29, change from baseline to Week 52, mean (SD)§ Physical Impact Score‡ 3.0 (13.5) −1.0 (11.8) 1.4 (13.5) 0.0008 0.1284 Psychological Impact Score 0.6 (14.4) −1.8 (15.8) −0.5 (15.3) 0.0683 0.4338 EQ-Visual Analogue Scale, change from −1.8 (13.2) 2.9 (13.3) 1.0 (12.8) <0.0001 0.0149 baseline to Week 52, mean (SD)§ EQ-5D Summary Health Index, change from −0.04 (0.20) 0.01 (0.18) −0.02 (0.20) 0.0091 0.3538 baseline to Week 52, mean (SD)§ SF-12, change from baseline to Week 52, mean (SD)§ Physical component −0.4 (7.0) 1.2 (7.3) 0.5 (7.3) 0.0116 0.1018 Mental component −1.4 (9.2) 0.7 (9.6) −0.1 (8.6) 0.0118 0.2342 ARR, annualized relapse rate; MSIS, Multiple Sclerosis Impact Scale; Gd+, gadolinium-enhancing. *P values estimated from a negative binomial regression model adjusted for number of relapses in 1-year period prior to study entry, baseline EDSS (≤2.5 vs. >2.5), and age (≤35 vs. >35). †P value estimated from Cox-proportional hazards model adjusting for number of relapses in 1-year prior to entry, baseline EDSS, and age. ‡Lower scores indicate improvement. §P value calculated using analysis of covariance for difference between treatment groups, controlling for baseline score. ¶MRI substudy, N = 307 (placebo, n = 104; DAC HYP 150 mg, n = 101; DAC HYP 300 mg, n = 102). ∥P value estimated from a negative binomial model adjusted for the baseline number of Gd+ lesions. **P value estimated from a negative binomial model adjusted for the baseline number of T2 lesions. ††P value estimated from Cox-proportional hazards model adjusting for baseline EDSS and age. ΔP value estimated from an analysis of covariance model adjusted for baseline EDSS. ‡‡P value estimated from a proportional odds model adjusted for the baseline number of Gd+ lesions. ∞P-value based on analysis of covariance on ranked data adjusted for baseline normalized brain volume.

MRI.

DAC HYP reduced new MS lesion activity, as defined by MRI, in both the entire study population and a subset with monthly MRIs performed between weeks 8 to 24 (Table 36). In contrast to the clinical endpoints, the point estimates of efficacy were marginally stronger in the 300 mg dose group compared to the 150 mg dose group even after adjustment for the potential baseline imbalances. Longitudinal analysis demonstrated that Gd+ lesion activity was higher in the 150 mg dose group compared to the 300 mg dose group in the first few months of treatment but was similar by week 52. (Table 36). Sensitivity analyses that included the 21 patients from the one excluded study site yielded similar results for all efficacy analyses.

Safety.

Adverse events (AEs) occurred in a similar proportion of patients in the DAC HYP 150 mg (73%), DAC HYP 300 mg (76%) and placebo (79%) groups (Table 37). Serious AEs, occurred in 26% of the placebo, 15% in the DAC HYP 150 mg and 17% in the DAC HYP 300 mg groups. Excluding MS relapses, SAEs occurred in 6%, 7% and 9% of patients in each group (Table 37). AEs that occurred in >5% of DAC HYP patients are shown in Table 37. The incidence of serious infections was 2% in DAC HYP-treated patients versus 0% in placebo. Among the 7 patients who had a serious infection while dosing was ongoing, 1 discontinued treatment due to the serious infection and 6 restarted treatment after the infection resolved. The incidence of cutaneous events was 18% in the DAC HYP 150 mg, 22% in the DAC HYP 300 mg, and 13% in the placebo groups (Table 37). Serious cutaneous events occurred in 1% of DAC HYP-treated patients. One DAC HYP-treated patient who was recovering from a serious rash died due to a complication of a psoas abscess. At autopsy, a psoas abscess, which had been previously undiagnosed, was found to involve a mesenteric artery and had resulted in local thrombosis and acute ischemic colitis. Five malignancies occurred during the trial: two cases of cervical carcinoma (1 each in the placebo and DAC HYP 150 mg group); one case of thyroid neoplasm in the DAC HYP 150 mg group was a non-serious thyroid nodule; and two cases of melanoma in the DAC HYP 300 mg group. The cases of melanoma were treated with local excision without reported recurrence.

TABLE 37 Adverse Events Summary DAC HYP DAC HYP Placebo (n = 204) 150 mg (n = 208) 300 mg (n = 209) Adverse event summary Any adverse event, n (%) 161 (79) 151 (73) 159 (76) Any serious adverse event, n (%) 53 (26) 32 (15) 36 (17) Any serious adverse event excluding 12 (6) 15 (7) 19 (9) MS relapse, n (%) Death, n 0 1* 0 Common Adverse Events that Occurred in ≥5% of DAC HYP Patients During Treatment MS Relapse, n (%) 76 (37) 43 (21) 41 (20) Nasopharyngitis, n (%) 31 (15) 30 (14) 29 (14) Upper respiratory tract infection, n (%) 14 (7) 18 (9) 21 (10) Headache, n (%) 20 (10) 18 (9) 20 (10) Pharyngitis, n (%) 8 (4) 13 (6) 13 (6) Adverse events of interest Infections, n (%) 89 (44) 104 (50) 112 (53) Serious Infections, n (%) 0 6 (3) 3 (1) Cutaneous events, n (%) 27 (13) 38 (18) 45 (22) Injection-site reaction, erthyma, 3 (1) 4 (2) 4 (2) Severe cutaneous events, n (%) 0 2 (<1) 2 (1) Malignancy, n (%) 1 (<1) 2 (<1) 2 (<1) Incidence of ALT Abnormalities 1-3x ULN, n (%) 64 (31) 54 (26) 62 (30) 3-5x ULN, n (%) 6 (3) 7 (3) 6 (3) >5x ULN, n (%) 1 (<1) 9 (4) 8 (4) ULN, upper limit of normal; ALT, alanine aminotransferase

Laboratory Findings.

Patients treated with DAC HYP had an increase in total NK cell count (cells/mm³) compared with placebo at Week 52 (median: 42.0 (150 mg DAC HYP); 46.5 (300 mg DAC HYP); vs −4.5 placebo; p=<0.001). The increase in total NK cell numbers was related to a selective increase in CD56^(bright) NK cells from a median of 7.77 at baseline to 44.84 at end of treatment. In contrast, there were only marginal changes in CD56^(dim) NK cells (median changes from 122.68 to 123.70). Expansion of CD56^(bright) NK cells was apparent at the first post-baseline time point (Week 4) in both DAC HYP arms versus placebo (p<0.0001). CD56^(bright) NK cells expanded from a median of 0.6% of lymphocytes at baseline to 2.8% at Week 52. In contrast, patients treated with DAC HYP had a modest decrease in B-cell and total lymphocyte counts (Table 38). Both CD4⁺ and CD8⁺ T-cell counts decreased by approximately 7-10% at Week 52 in DAC HYP-treated patients and the CD4+/CD8⁺ ratio remained constant during treatment.

TABLE 38 Changes in Lymphocyte Cell Counts Over 52 Weeks DAC HYP Placebo (n = 179) 150 mg (n = 184) 300 mg (n = 186) Total lymphocytes, mean cells/mm³ (SD) Baseline 1420.9 (450.3) 1444.1 (441.9) 1395.7 (471.4) Week 24 1455.7 (468.7) 1380.9 (426.4) 1314.7 (366.3) % Change Week 24 4.81 (29.48) −1.30 (27.60) −1.2 (29.01) Week 52 1397.2 (414.4) 1332.5 (398.4) 1286.2 (446.8) % Change Week 52 1.89 (26.60) −3.63 (27.95) −3.69 (27.74) B cells, mean cells/mm³ (SD) Baseline 174.4 (91.9) 175.5 (89.8) 167.2 (89.6) Week 24 187.4 (110.3) 169.4 (107.2) 150.9 (94.9) % Change Week 24 17.77 (69.77) −1.19 (42.49) −3.35 (53.29) Week 52 177.2 (81.2) 157.2 (92.9) 141.0 (76.9) % Change Week 52 12.57 (47.25) −4.25 (43.67) −11.26 (38.15) NK cells, mean cells/mm³ (SD) Baseline 163.4 (76.7) 166.2 (110.9) 175.0 (95.7) Week 24 169.0 (80.3) 199.3 (106.6) 205.1 (87.9) % Change Week 24 9.50 (44.88) 32.71 (56.55) 33.03 (65.13) Week 52 158.3 (82.7) 213.0 (113.9) 223.5 (117.9) % Change Week 52 2.77 (43.07) 48.14 (82.09) 50.5 (84.1) CD4+ cells, mean cells/mm³ (SD) Baseline 686.9 (248.3) 698.8 (241.3) 664.4 (261.0) Week 24 692.8 (235.3) 646.2 (216.2) 606.5 (222.8) % Change Week 24 3.87 (26.72) −4.42 (27.20) −4.66 (28.24) Week 52 682.9 (219.6) 612.8 (202.6) 581.7 (259.8) % Change Week 52 2.77 (26.45) −6.95 (30.33) −8.94 (29.97) CD8+ cells, mean cells/mm³ (SD) Baseline 355.3 (156.2) 361.4 (146.4) 353.4 (165.8) Week 24 363.9 (176.8) 328.0 (139.6) 309.5 (133.4) % Change Week 24 4.11 (40.70) −4.73 (30.67) −6.49 (30.23) Week 52 344.9 (160.9) 315.7 (139.6) 295.7 (151.0) % Change Week 52 3.05 (44.60) −9.12 (30.96) −9.83 (33.94) CD4/CD8 ratio Baseline 2.2 (0.9) 2.1 (0.9) 2.1 (09) Week 24 2.2 (0.9) 2.2 (0.9) 2.2 (1.0) % Change Week 24 4.23 (25.38) 2.87 (18.55) 4.07 (19.85) Week 52 2.3 (1.0) 2.2 (0.9) 2.2 (0.9) % Change Week 52 6.36 (30.00) 4.57 (18.77) 4.47 (20.63) SD, standard deviation; NK, natural killer.

Liver function test (LFT) abnormalities were above 5×ULN, occurred in 4% of DAC- and <1% of placebo-treated patients. These abnormalities typically occurred late in the treatment period (median onset+day 308) and resolved with a median time of 62 days. Of the 17 DAC HYP-treated patients with elevations of >5×ULN, 6 continued or resumed treatment with DAC HYP for at least 6 months after resolution, all without recurrence during this period. In 2 patients, LFT elevations were associated with infections (one case of hepatitis B and one case of cytomegalovirus infection).

Immunogenicity.

At week 24, neutralizing antibodies to DAC HYP were detected in 6 (2%) DAC HYP-treated patients (5 patients in the 150 mg dose group and 1 subject in the 300 mg dose group). In some patients these antibodies were transient, and at week 52 neutralizing antibodies to DAC HYP were present in only 1 subject from each DAC HYP dose group.

7.10. DAC Compositions are Useful to Treat Non-Relapsing Forms or Stages of MS and Progressive Forms of MS

In the SELECT study, three-month confirmed disability was defined as a 1.0-point or more increase in Expanded Disability Status Scale (EDSS) score for patients with a baseline EDSS ≥1.0 or a 1.5-point or more increase for patients with a baseline EDSS=0 between baseline and Week 52. Sustained disability progression could begin at any visit. Confirmation of the progression had to occur at least 12 weeks later at a visit when a relapse was not occurring. Disability relapses were defined as a relapse that occurred within 30 days prior to the onset of disability progression.

Disability progression occurred in 13% (n=25) of patients treated with placebo and in 6% (n=26) of patients treated with DAC HYP (Risk Reduction=50% [95% confidence interval: 15%-70%]; p=0.009). The median EDSS score of patients who progressed was 2.5 in both placebo- and DAC HYP-treated groups at baseline and 4.0 in both groups at the time that disability progression was confirmed. The effect of DAC HYP disability progression was mediated similarly by a reduction in the proportion of patients with disabling relapses in the placebo-treated (6%; n=11) versus DAC HYP-treated (2%; n=10) groups and by a reduction in disability progression that was independent of relapse (7% [n=4] of placebo-treated patients versus 4% [n=16] of DAC HYP-treated patients). The observation that disability progression is reduced by DAC HYP in a manner that is independent of relapse, i.e., in relapse-independent disease progression, supports the use of DAC therapy in patients who do not suffer relapses or exacerbations for significant periods of time, for example at least 6 months, 12 months, 18 months or even longer and/or who suffer from progressive forms of MS that are characterized by disease progression without relapses or exacerbations, such as primary progressive, secondary progressive and other progressive forms of MS.

While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the invention(s). 

1. A method of treating a human subject suffering from a progressive form of multiple sclerosis, comprising subcutaneously administering to the subject about 150 mg to about 300 mg of a daclizumab once every four weeks for a period of at least one year. 2-11. (canceled) 